Reduced and oxidized polysaccharides and methods of use thereof

ABSTRACT

The present invention is directed to reduced and highly oxidized polysaccharides, such as alginates, that are useful for encapsulating therapeutic or diagnostic agents, or lipid based nanoparticles, e.g., liposomes or virosomes, encapsulating therapeutic or diagnostic agents, prior to their delivery into a subject, as well as methods for making and using them.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/770,937, filed on Apr. 25, 2018; which is a 35 U.S.C. § 371national stage filing of International Application No.PCT/US2016/058866, filed on Oct. 26, 2016; which claims priority to U.S.Provisional Application No. 62/246,462, filed on Oct. 26, 2015 and U.S.Provisional Application No. 62/351,162, filed on Jun. 16, 2016. Theentire contents of each of the foregoing applications are herebyincorporated herein by reference.

BACKGROUND OF THE INVENTION

A hydrogel is a polymer gel comprising a network of crosslinked polymerchains. The network structure of hydrogels allows them to absorbsignificant amounts of water. Some hydrogels are highly stretchable andelastic; others are viscoelastic. Hydrogels have been used fortherapeutic applications, e.g., as vehicles for in vivo delivery oftherapeutic agents, such as small molecules, cells and biologics.

Hydrogels are commonly produced from polysaccharides, such as alginates.The polysaccharides may be chemically manipulated to modulate theirproperties and properties of the resulting hydrogels. For example,oxidizing a polysaccharide by reacting it with an oxidizing agent thatconverts alcohols in the polysaccharide into aldehydes, significantlyincreases the biodegradability of the resulting hydrogel. However, theoxidation of polysaccharides is also associated with undesirable sideeffects. For example, the aldehydes produced by oxidation can react withamino groups present on proteins or other molecules, causing in vivotoxicity and/or damage to therapeutic agents (cargo) that may beencapsulated by hydrogels produced from the oxidized polysaccharides.The aldehydes present in an oxidized polysaccharide can also react withvarious chemical moieties that may be present in the vicinity of thepolysaccharide, such as click reagents, resulting in their degradation.Accordingly, there is a need in the art for biodegradablepolysaccharides that may be used for preparing hydrogels that arenon-toxic, non-reactive and biodegradable. There is also a need in theart for polysaccharides that may be used to prepare hydrogels with asufficiently small pore size for encapsulating therapeutic agents ofsmall molecular weights. There is also a need in the art forpolysaccharides that may be used to prepare hydrogels capable ofencapsulating and retaining liposomes and delivering the liposomes to adesired location in vivo.

SUMMARY OF THE INVENTION

The present invention provides compositions comprising certain reducedand certain highly oxidized polysaccharides. It has been surprisinglydiscovered that these reduced and highly oxidized polysaccharides areparticularly well suited for producing hydrogels that may be used forencapsulating therapeutic or diagnostic agents or for encapsulatinglipid based nanoparticles, e.g., liposomes or virosomes, used forencapsulating therapeutic or diagnostic agents, for delivery to asubject. The hydrogels of the invention produced using the reducedand/or highly oxidized polysaccharides are biodegradable and less toxicand reactive than the hydrogels previously known in the art.

Accordingly, in some embodiments, the present invention provides acomposition comprising a reduced polysaccharide, wherein the reducedpolysaccharide comprises less than 2% of residual aldehydes.

In some embodiments, the present invention also provides a compositioncomprising a reduced polysaccharide, wherein the reduced polysaccharidecomprises less than 3% of residual aldehydes; wherein the reducedpolysaccharide is produced by reacting a polysaccharide with a diolspecific oxidizing agent, thereby producing an oxidized polysaccharidecomprising 0.1% or more oxidation on a molar basis; followed by reactingsaid oxidized polysaccharide with a water soluble aldehyde specificreducing agent to produce the reduced polysaccharide.

In some aspects, the reduced polysaccharide is produced by reacting apolysaccharide with a diol specific oxidizing agent, thereby producing adiol containing oxidized polysaccharide comprising 0.1% or moreoxidation on a molar basis; followed by reacting said oxidizedpolysaccharide with a water soluble aldehyde specific reducing agent toproduce said reduced polysaccharide.

In some aspects, also provided is a composition comprising a reducedpolysaccharide, wherein the reduced polysaccharide is produced byreacting a polysaccharide with a diol specific oxidizing agent, therebyproducing a diol containing oxidized polysaccharide; followed byreacting the oxidized polysaccharide with a water soluble aldehydespecific reducing agent to produce said reduced polysaccharide. In someaspects, the polysaccharide comprises less than 15% residual aldehydes,e.g., 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%or 0.1%.

In some embodiments, the polysaccharide is selected from the groupconsisting of alginate, agarose, pullulan, scleroglucan, chitosan,elsinan, xanthan gum, dextran, mannose, gellan, levan, cellulose,hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C,chondroitin sulfate E and β-d-glucans. In one embodiment, thepolysaccharide is alginate. In one embodiment, the reducedpolysaccharide comprises algoxinol having the following structure:

In some aspects, the aldehyde specific water soluble reducing agent isselected from the group consisting of sodium borohydride (NaBH₄); sodiumcyanoborohydride (NaCNBH₃); hydrogen gas (H₂) in the presence of acatalyst; ammonia borane (H₃NBH₃); and a borane complex. In a specificembodiment, the aldehyde specific water soluble reducing agent isammonia borane. In another specific embodiment, the diol specificoxidizing agent is sodium periodate.

In some embodiments, the composition further comprises a cross-linkingagent attached to the reduced polysaccharide. In some aspects, thecross-linking agent is a click reagent, e.g., a click reagent isselected from the group consisting of azide, dibenzocyclooctyne (DBCO),transcyclooctene, tetrazine and norbornene and variants thereof.

In some embodiments, the present invention also provides a compositioncomprising a reduced polysaccharide and a cross-linking agent attachedto the reduced polysaccharide. In some aspects, the polysaccharide is analginate polymer. In some embodiments, the reduced polysaccharidecomprises algoxinol having the following structure:

In some embodiments, the reduced polysaccharide is produced by a methodcomprising the steps of reacting the polysaccharide with a diol specificoxidizing agent to produce an aldehyde containing oxidizedpolysaccharide; and reacting the oxidized polysaccharide with a watersoluble aldehyde specific reducing agent to produce the reducedpolysaccharide.

In some embodiments, the diol specific oxidizing agent is sodiumperiodate. In some embodiments, the water soluble aldehyde specificreducing agent is ammonia borane.

In some aspects, provided is a composition comprising a highly oxidizedpolysaccharide and a cross-linking agent attached to the highly oxidizedpolysaccharide. In some embodiments, the polysaccharide is an alginatepolymer. In some aspects, the highly oxidized polysaccharide comprisesalgoxalate having the following structure:

In some embodiments, the highly oxidized polysaccharide is produced by amethod comprising the steps of reacting the polysaccharide with a diolspecific oxidizing agent to produce an aldehyde containing oxidizedpolysaccharide; and reacting the oxidized polysaccharide with a secondoxidizing agent which converts aldehydes into carboxylic acids toproduce said highly oxidized polysaccharide.

In one aspect, the diol specific oxidizing agent is sodium periodate. Inanother aspect, the second oxidizing agent is sodium chlorite.

In some embodiments, the cross-linking agent is a click reagent, e.g., aclick reagent selected from the group consisting of azide,dibenzocyclooctyne (DBCO), transcyclooctene, tetrazine and norborneneand variants thereof.

In some embodiments, the present invention also provides a hydrogelcomprising the compositions of the invention.

In some aspects, the hydrogel may further comprise a therapeutic agent,e.g., selected from the group consisting of a cell, a small molecule anda biologic. In some embodiments, the biologic is selected from the groupconsisting of a peptide, a DNA molecule, an RNA molecule, and a PNAmolecule. In one embodiment, the biologic is a peptide, e.g., anangiogenesis factor, e.g., selected from the group consisting of FGF,VEGF, VEGFR, IGF, NRP-1, Ang1, Ang2, PDGF, PDGFR, TGF-β, endoglin, aTGF-β receptor, MCP-1, integrin, VE-cadherin, CD31, ephrin, plasminogenactivator, plasminogen activator inhibitor-1, eNOS, COX-2, AC133, ID1,ID3, and HGF. In a specific aspect, the angiogenesis factor is VEGF.

In some embodiments, the hydrogel of the invention further comprises acell, e.g., a mammalian cell, such as a human mesenchymal stem cell(hMSC).

In some aspects, the present invention provides a hydrogel comprising aplurality of reduced and/or oxidized polysaccharides cross-linked toeach other, wherein the hydrogel comprises a mesh size of about 10 nm orless, e.g., about 10 nm, about 9 nm, about 8 nm, about 7 nm, about 6 nm,about 5 nm, about 4 nm, about 3 nm, about 2 nm or about 1 nm or less.

In some embodiments, the oxidized polysaccharides are highly oxidizedalginate polymers. In some embodiments, the highly oxidized alginatepolymers are produced by a method comprising the steps of reacting analginate with a diol specific oxidizing agent to produce an aldehydecontaining oxidized alginate; and reacting the aldehyde containingoxidized alginate with a second oxidizing agent which converts aldehydesinto carboxylic acids, thereby producing said highly oxidized alginatepolymers.

In one embodiment, the diol specific oxidizing agent is sodiumperiodate. In another embodiment, the second oxidizing agent is sodiumchlorite.

In some embodiments, the oxidized alginate polymers are about 0.1% 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or 99% oxidized.

In some embodiments, the reduced polysaccharides are reduced alginatepolymers. In some embodiments, the reduced alginate polymers areproduced by a method comprising the steps of reacting an alginate with adiol specific oxidizing agent to produce an aldehyde containing oxidizedalginate; and reacting the aldehyde containing oxidized alginate with awater soluble aldehyde specific reducing agent, thereby producing saidreduced alginate polymers.

In one embodiment, the diol specific oxidizing agent is sodiumperiodate. In another embodiment, the water soluble aldehyde specificreducing agent is ammonia borane.

In some aspects, the hydrogel of the invention comprises an average meshsize of about 10 nm to about 1 nm, about 10 nm to about 4 nm, about 7 nmto about 3 nm, about 5 nm to about 1 nm, about 4 nm to about 2 nm, orabout 3 nm to about 0.5 nm.

In some aspects, the hydrogel of the invention further comprises atherapeutic or diagnostic agent, e.g., a cell, a small molecule and abiologic. In some aspects, the biologic is selected from the groupconsisting of a peptide, a DNA molecule, an RNA molecule, and a PNAmolecule.

In some embodiments, the biologic is a peptide. In some embodiments, thepeptide has a molecular weight of 500 kDa or less, e.g., about 450 kDaor less, about 300 kDa or less, about 150 kDa or less, about 100 kDa orless, about 50 kDa or less, about 25 kDa or less or about 10 kDa orless.

In some aspects, the peptide is an angiogenesis factor, e.g., FGF, VEGF,VEGFR, NRP-1, Ang1, Ang2, PDGF, PDGFR, IGF, TGF-β, endoglin, a TGF-βreceptor, MCP-1, integrin, an integrin ligand, VE-cadherin, CD31,ephrin, plasminogen activator, plasminogen activator inhibitor-1, eNOS,COX-2, AC133, ID1, ID3, and HGF. In a specific embodiment, theangiogenesis factor is VEGF.

In some embodiments the cell is a mammalian cell, e.g., a humanmesenchymal stem cell (hMSC).

In some embodiments, the polysaccharides comprise a cross-linking agentattached to the polysaccharides. In some aspects, the cross-linkingagent is a click reagent, e.g., azide, dibenzocyclooctyne,transcyclooctene, tetrazine and norbornene and variants thereof.

In some embodiments, the present invention also provides an implantableor injectable device comprising the hydrogel of the invention.

In some aspects, the present invention also provides a method ofproducing a reduced polysaccharide, the method comprising the steps ofreacting a polysaccharide with a diol specific oxidizing agent toproduce an aldehyde containing oxidized polysaccharide; and reacting thealdehyde containing oxidized polysaccharide with an aldehyde specificwater soluble reducing agent, thereby producing the reducedpolysaccharide.

In some embodiments, the aldehyde specific water soluble reducing agentis sodium borohydride (NaBH₄); sodium cyanoborohydride (NaCNBH₃);hydrogen gas (H₂) in the presence of a catalyst, e.g., a nickel (Ni), aplatinum (Pl) or a palladium (Pd) catalyst; ammonia borane (H₃NBH₃); ora borane complex, e.g., a bis-carbonate borane complex ([(BH₃)₂CO₂]²⁻.2Na⁺), a borane dimethylamine complex [(CH₃)₂NH.BH₃]; a boranetert-butylamine complex [(CH₃)₃CNH₂.BH₃]; or a borane-pyrimidinecomplex. In a specific embodiment, the aldehyde specific water solublereducing agent is ammonia borane. In another specific embodiment, thediol specific oxidizing agent is sodium periodate.

In some embodiments, the method further comprises reacting saidpolysaccharide with a cross-linking agent. In some aspects, thecross-linking reagent is a click reagent, e.g., azide,dibenzocyclooctyne (DBCO), transcyclooctene, tetrazine and norborneneand variants thereof. In a specific embodiment, the polysaccharide is analginate polymer. In an embodiment, the alginate polymer is of amolecular weight of less than 500 kDa. In an embodiment, the reducedpolysaccharide comprises algoxinol having the following structure:

In some aspects, the present invention also provides a method ofproducing a highly oxidized polysaccharide covalently attached to across-linking agent, the method comprising the steps of reacting apolysaccharide with a diol specific oxidizing agent to produce analdehyde containing oxidized polysaccharide; reacting the aldehydecontaining oxidized polysaccharide with a second oxidizing agent whichconverts aldehydes into carboxylic acids, thereby producing the highlyoxidized polysaccharide; and reacting the polysaccharide with across-linking agent.

In some aspects, the second oxidizing agent is selected from the groupconsisting of sodium chlorite, bromine, dilute nitric acid (NHO₃),silver oxide, a copper(II) complex, potassium permanganate (KMnO₄) andhydrogen peroxide (H₂O₂). In a specific embodiment, the second oxidizingagent is sodium chlorite.

In some embodiments, the cross-linking agent is a click reagent, e.g.,azide, dibenzocyclooctyne (DBCO), transcyclooctene, tetrazine andnorbornene and variants thereof.

In some aspects, the polysaccharide is an alginate polymer. In oneaspect, the alginate polymer has a molecular weight of less than 500kDa.

In one embodiment, the highly oxidized polymer comprises algoxalatehaving the following structure:

The present invention also provides a drug delivery compositioncomprising a lipid based nanoparticle encapsulating a therapeutic ordiagnostic agent; and the hydrogel of the invention encapsulating thelipid based nanoparticle. In an embodiment, the lipid based nanoparticleis a liposome or a virosome.

In some embodiments, the lipid based nanoparticle remains encapsulatedin the hydrogel for at least 1 day, at least 10 days, at least 15 days,at least 20 days, at least 25 days, at least 30 days, at least 35 days,at least 40 days, at least 45 days, at least 50 days, at least 55 days,at least 60 days, at least 65 days, at least 70 days, at least 75 daysor at least 80 days.

In some aspects, the therapeutic or diagnostic agent is selected fromthe group consisting of a cell, a small molecule and a biologic. In someaspects, the biologic is selected from the group consisting of apeptide, an antibody or a fragment thereof, a vaccine, a DNA molecule,an RNA molecule, and a PNA molecule. In some aspects, the therapeutic ordiagnostic agent is selected from the group consisting of a STINGadjuvant, a CRISPR-Cas 9 reagent and an adjuvant-loaded subcellularvesicle derived from disrupted cancer cells. In some aspects, thetherapeutic or diagnostic agent is a vaccine.

In some aspects, the present invention also provides a drug deliverycomposition, comprising a lipid based nanoparticle encapsulating atherapeutic or diagnostic agent; and a click-conjugated polymer hydrogelencapsulating the lipid based nanoparticle, wherein the click-conjugatedpolymer hydrogel comprises a biodegradable polymer; and wherein thepolymer is not susceptible to degradation by a host endogenous enzyme.In an embodiment, the lipid based nanoparticle is a liposome or avirosome.

In some embodiments, the drug delivery composition allows a sustaineddelivery of intact lipid based nanoparticle to a desired location in thehost.

In some embodiments, the polysaccharide is selected from the groupconsisting of alginate, agarose, pullulan, scleroglucan, chitosan,elsinan, xanthan gum, dextran, mannose, gellan, levan, cellulose,hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C,chondroitin sulfate E and β-d-glucans. In a specific embodiment, thepolysaccharide is alginate.

In some aspects, the click-conjugated polymer hydrogel is generated byuse of at least one click reagent selected from the group consisting ofazide, dibenzocyclooctyne, transcyclooctene, tetrazine and norborneneand variants thereof.

In some embodiments, the alginate comprises a degree of clicksubstitution of about 0.01% to about 99%, e.g., about 0.01% to about0.05%, about 0.02% to about 0.1%, about 0.05% to about 0.5%, about 0.1%to about 1%, about 0.5% to about 15%, about 5% to about 10%, about 2% toabout 20%, about 15% to about 40%, about 30% to about 50%, about 40% toabout 70%, about 50% to about 80% or about 60% to about 99%. In someaspects, the click-conjugated polymer hydrogel comprises alginate thatis about 0.1% to about 99% oxidized, e.g., about 0.1% to about 0.5%,about 0.2% to about 1%, about 0.5% to about 10%, about 5% to about 20%,about 15% to about 40%, about 25% to about 50%, about 40% to about 70%,about 50% to about 80% or about 75% to about 99% oxidized.

In some aspects, the click-conjugated alginate hydrogel has beenprepared from alginate present at a concentration of about 1% to about99% w/v, e.g., about 1% to about 5%, about 2% to about 10%, about 5% toabout 20%, about 10% to about 30%, about 20% to about 50%, about 40% toabout 70%, about 60% to about 80% or about 75% to about 99% w/v. In someembodiments, the lipid based nanoparticle remains encapsulated in theclick-conjugated polymer hydrogel for at least 1 days, at least 10 days,at least 15 days, at least 20 days, at least 25 days, at least 30 days,at least 35 days, at least 40 days, at least 45 days, at least 50 days,at least 55 days, at least 60 days, at least 65 days, at least 70 days,at least 75 days or at least 80 days.

In some aspects, the present invention also provides a drug deliverycomposition, comprising a lipid based nanoparticle encapsulating atherapeutic or diagnostic agent; and an alginate hydrogel encapsulatingthe lipid based nanoparticle, wherein the hydrogel comprises alginatecomprising about 50% or less oxidation on a molar basis, e.g., about 45%or less, about 40% or less, about 35% or less, about 30% or less, about25% or less, about 20% or less, about 15% or less, about 10% or less,about 5% or less, about 1% or less, about 0.5% or less or about 0.1% orless. In an embodiment, the lipid based nanoparticle is a liposome or avirosome. In one aspect, composition allows a sustained delivery ofintact lipid based nanoparticle to a desired location in a host.

In some aspects, the click-conjugated polymer hydrogel is generated byuse of at least one click reagent selected from the group consisting ofazide, dibenzocyclooctyne, transcyclooctene, tetrazine and norborneneand variants thereof. In some aspects, the alginate is conjugated to aclick reagent and wherein the alginate comprises a degree of clicksubstitution of about 0.01% to about 90%.

In some aspects, the click-conjugated polymer hydrogel comprisesalginate that is about 0.1% to about 99% oxidized.

In some embodiments, the alginate hydrogel has been prepared fromalginate present at the concentration of about 1% to about 80% w/v. Insome embodiments, the lipid based nanoparticle remains encapsulated inthe hydrogel for at least 1 days, at least 10 days, at least 15 days, atleast 20 days, at least 25 days, at least 30 days, at least 35 days, atleast 40 days, at least 45 days, at least 50 days, at least 55 days, atleast 60 days, at least 65 days, at least 70 days, at least 75 days orat least 80 days.

In some aspects, the therapeutic or diagnostic agent is selected fromthe group consisting of a cell, a small molecule and a biologic. In someembodiments, the biologic is selected from the group consisting of apeptide, an antibody or a fragment thereof, a vaccine, a DNA molecule,an RNA molecule, and a PNA molecule. In some embodiments, the RNAmolecule is selected from the group consisting of an mRNA, an RNAi, ansiRNA, an shRNA, a microRNA, an isRNA, a lncRNA and an antisense RNA.

In some aspects, the therapeutic or diagnostic agent is selected fromthe group consisting of a STING adjuvant, a CRISPR-Cas 9 reagent and anadjuvant-loaded subcellular vesicle derived from disrupted cancer cells.

In some embodiments, the liposome is a cationic liposome. In someaspects, the desired location is the cytosol of a cell.

In some embodiments, the present invention also provides a method fortreating a subject in need thereof, the method comprising administeringto the subject an effective amount of the hydrogel, the implantable orinjectable device or the drug delivery composition of the invention,thereby treating said subject.

In some aspects, the subject is suffering from a disease or a conditionselected from the group consisting of ischemia, an eye related disorderor an ear related disorder.

In some embodiments, the present invention also provides a method oftreating chronic ischemia or enhancing engraftment of a transplantedtissue in a subject in need thereof, the method comprising administeringto said subject an effective amount of the hydrogel, the implantable orinjectable device or the drug delivery composition of the invention,thereby treating said chronic ischemia or enhancing engraftment of saidtransplanted tissue in said subject.

In some aspects, the hydrogel or the implantable or injectable device orthe drug delivery composition is administered locally, e.g., to a siteof ischemia or to the tissue to be engrafted before and/or aftertransplantation.

In some aspects, the hydrogel or the implantable or injectable device orthe drug delivery composition comprises a peptide at a dose that is atleast about 10 times smaller, e.g., about 15 times smaller, about 20times smaller, about 50 times smaller, than the recommended dose forsaid peptide when said peptide is delivered in soluble form.

In a specific aspect, the peptide is VEGF and IGF. In an embodiment, thesubject is a human.

The present invention is further illustrated by the following detaileddescription and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is a schematic showing conjugation of click reagent tounoxidized alginate.

FIG. 1b is a schematic showing conjugation of click reagent to oxidizedalginate, illustrating the formation of an unstable (hydrolyzable) iminebond, which is detrimental to further covalent click cross-linking.

FIG. 1c is a schematic showing conjugation of click reagent to highlyoxidized alginate, illustrating the additional carboxylic acid groupsproduced that are available for click conjugation.

FIG. 2a is a bar graph showing the loss of VEGF bioactivity, asdetermined by ELISA, as a function of exposure to oxidized alginates.

FIG. 2b is a picture of vials containing the oxidized alginatesconjugated to Tz and Nb after incubation, illustrating the apparentchange in click conjugates due to the presence of aldehydes in oxidizedalginates.

FIG. 3a is a chemical scheme showing oxidation of alginate to produceoxidized alginate containing reactive aldehydes; and subsequentselective reduction or oxidation of the oxidized alginate in order toeliminate the reactive aldehydes.

FIG. 3b is a bar graph showing % residual oxidation in oxidizedalginates that have been further reduced with ammonia borane or sodiumborohydride, or that have been further oxidized with sodium chlorite.

FIG. 4a is a bar graph showing % change in VEGF bioactivity measured byELISA after 5 days of incubation in EBM, illustrating the loss of VEGFbioactivity as a function of exposure to oxidized or reduced alginate.

FIG. 4b is a bar graph showing % change in VEGF bioactivity measured byELISA after 5 days of incubation in 1% BSA solution, illustrating theloss of VEGF bioactivity as a function of exposed to oxidized alginates.

FIG. 5a is a panel of two pictures of vials containing HighOx alginatesconjugated to Tz and Nb at day 0 (top panel) and HighOx Sc alginatesconjugated to Tz and Nb at day 0 (bottom panel), illustrating theapparent change in click conjugates due to the presence of aldehydes inoxidized alginates and the lack of change in the conjugates containingalgoxinol and algoxalate.

FIG. 5b is a bar graph showing relative amounts of Tz present in HighOxand HighOx SC alginates as measured by NMR, illustrating the additionalpotential for click conjugation of the algoxalate containing alginatevia the additional carboxylic acid moieties.

FIG. 6a is a UV-Vis spectrum of Tz-conjugated HighOx alginate materialwith 20-50% oxidation at day 28 (top panel) and a UV-Vis spectrum ofTz-conjugated HighOx alginate material treated with sodium chloritecontaining 20% and 30% conjugation at day 14. FIG. 6a illustrates thechange in Tz UV-Vis spectrum caused by the reaction of the clickmoieties with alginate aldehydes present in the oxidized alginatematerial.

FIG. 6b is a panel of two pictures of vials containing Tz-conjugatedHighOx alginate material treated with ammonia borane and the HighOxalginate materials treated with sodium chlorite. FIG. 6b illustrates thechange in the color of the Tz solution caused by the reaction of clickmoieties with alginate aldehydes present in the oxidized alginatematerial.

FIG. 6c is a UV-Vis spectrum of Nb-conjugated HighOx alginate materialwith 20-50% oxidation at day 28 (top panel) and a UV-Vis spectrum ofNb-conjugated HighOx alginate material treated with sodium chlorite,containing 20%-50% conjugation at day 14. FIG. 6c illustrates the changein Nb UV-Vis spectrum caused by the reaction of the click moieties withalginate aldehydes present in the oxidized alginate material.

FIG. 6d is a panel of two pictures of vials containing Nb-conjugatedHighOx alginate material with 20-50% oxidation at day 28 (top panel) andNb-conjugated HighOx alginage material treated with sodium chloritecontaining 20%-50% conjugation at day 14 (bottom panel). FIG. 6dillustrates the change in the color o the Nb solution caused by thereaction of click moieties with ainate aldehydes present in the oxidizedalginate material.

FIG. 6e is an NMR spectrum of Nb-conjugated alginate material that hasbeen oxidized with sodium periodate, showing the broadening of Nb peaksas well as the presence of aldehyde peaks.

FIG. 6f is an NMR spectrum of Tz-conjugated alginate material that hasbeen oxidized with sodium periodate, showing the broadening of Nb peaksand the presence of aldehyde peaks.

FIG. 6g is an NMR spectrum of Nb-conjugated alginate material that hasbeen oxidized with sodium periodate and then further oxidized withsodium chlorite, showing the lack of broadening of Nb peaks and theabsence of aldehyde peaks.

FIG. 6h is an NMR spectrum of Tz-conjugated alginate material that hasbeen oxidized with sodium periodate and then further oxidized withsodium chlorite, showing the lack of broadening of Nb peaks and theabsence of aldehyde peaks..

FIG. 6i is a representative FTIR spectrum of the alginate material,demonstrating the production of carboxylic acid peaks following thetreatment with sodium chlorite.

FIG. 6j is a UV-Vis spectra for Tz-conjugated and Nb-conjugated alginatematerials at day 15, showing the lack of color change for clickconjugated alginates containing algoxinol and algoxalate.

FIG. 6k is an NMR spectra for the alginate material that has beenoxidized with sodium periodate and further oxidized with sodiumchlorite, showing the absence of aldehyde associated peaks (˜≥5.1 ppm).

FIG. 7a is a panel of two graphs showing degradation of Tz-conjugatedand Nb-conjugated MVG alginate (˜250 kDa starting MW) with 20% oxidation(left panel) and 50% oxidation (right panel), showing hydrolysis of thealginate polymer backbone as a function of time.

FIG. 7b is a panel of four graphs showing degradation of VLVG (30 kDastarting MW) alginate containing 0%-50% oxidation at 37° C. fornon-processed alginate (upper left panel), alginate reductivelyprocessed with ammonia borane (upper right panel), alginate reductivelyprocessed with sodium borohydride (lower left panel), and alginatereductively processed with ammonia borane (lower right panel)), showinghydrolysis of the alginate polymer backbone as a function of time.

FIG. 7c is a picture of vials containing 50% w/v solutions of MVG andVLVG materials with 20% oxidation and unoxidized VLVG at 50% w/v forcomparison. Unlike the parental alginate, the alginate materialcontaining algoxinol and algoxalate show increased solubility.

FIG. 8 is a graph showing UV-Vis absorption at 515 nm of Tz-conjugatedalginate material versus the conjugation equivalents.

FIG. 9 is a bar graph showing relative cell viability in the presence ofoxidized alginate containing 0-50% oxidation after 2-days of incubation.

FIG. 10 is a bar graph showing the upper limits of solubility (% w/v)for unoxidized alginate with molecular weight of 250 kDa (MVG, leftbar); unoxidized alginate with molecular weight of 30 kDa (VLVG, centerbar); and for VLVG material that was oxidized to 20%, and then furtheroxidized by using sodium chlorite and conjugated with clicks (Tz or Nb,right bar). FIG. 10 illustrates that, in constrast to the unoxidizedalginate, oxidized VLVG material is soluble to at least 50% w/v.

FIG. 11a is a bar graph showing the conjugation potential achieved forMVG and VLVG materials using Tz and Nb.

FIG. 11b is a graph showing the UV-Vis absorbance at 515 nm of the VLVGmaterial as a function of molar equivalents of Tz.

FIG. 12a is a graph showing the increase in the storage modulus(G′)versus time for VLVG materials at different degrees of substitution andconstant alginate concentration (% w/v).

FIG. 12b is a bar graph showing the maximal value (from FIG. 13a ) ofthe storage modulus (G′) for VLVG materials at different degrees ofsubstitution at the constant alginate concentration (% w/v).

FIG. 12c is a bar graph showing the value of mesh size for VLVGmaterials at different degrees of substitution at the constant alginateconcentration (% w/v), as derived from the maximal storage modulus (G′)depicted in FIG. 13 b.

FIG. 12d is a graph showing the increase in the storage modulus (G′)versus time for MVG material that was oxidized to 10% oxidation, reducedwith ammonia borane and then conjugated with Nb or Tz using 250 molarequivalents of Nb and Tz at the concentration of reduced alginate of 5%w/v, 10% w/v or 15% w/v.

FIG. 12e is a graph showing the increase in the storage modulus (G′)versus time for MVG material that was oxidized to 20% oxidation, reducedwith ammonia borane and then conjugated with Nb or Tz using 250 molarequivalents of Nb and Tz at the concentration of reduced alginate of 5%w/v, 10% w/v, 15% w/v or 20% w/v.

FIG. 12f is a graph showing the increase in the storage modulus (G′)versus time for LF 20/40 material that was oxidized to 20% oxidation,reduced with ammonia borane and then conjugated with Nb or Tz using 1000molar equivalents of Nb and Tz at the concentration of reduced alginateof 5% w/v, 10% w/v, 15% w/v or 20% w/v.

FIG. 13a is a graph showing cumulative release (in micrograms) offluorescein conjugated insulin from hydrogels produced usingclick-conjugated VLVG materials with different degrees of clickconjugation and constant alginate concentration (% w/v).

FIG. 13b is a graph showing the non-plateau region of the curve in FIG.14a corresponding to 2500 molar equivalents of Nb or Tz and 5%concentration of the Alg-N and Alg-T during hydrogel formation (2500 eq5% VLVG) and the linear fit.

FIG. 13c is a graph showing cumulative release (in micrograms) offluorescein conjugated BSA from hydrogels produced usingclick-conjugated VLVG materials with different degrees of clickconjugation and constant alginate concentration (% w/v).

FIG. 13d is a graph showing the non-plateau region of the curve in FIG.14c corresponding to 2500 molar equivalents of Nb or Tz and 5%concentration of the Alg-N and Alg-T during hydrogel formation (2500 eq5% VLVG) and the linear fit.

FIG. 13e is a graph showing cumulative release (in micrograms) offluorescein conjugated IgG from hydrogels produced usingclick-conjugated VLVG materials with different degrees of clickconjugation and constant alginate concentration (% w/v).

FIG. 13f is a graph showing the non-plateau region of the curve in FIG.14e corresponding to 2500 molar equivalents of Nb or Tz and 5%concentration of the Alg-N and Alg-T during hydrogel formation (2500 eq5% VLVG) and the linear fit.

FIG. 13g is a graph showing cumulative release (in micrograms) offluorescein conjugated insulin, BSA and IgG from hydrogels produced byCa²⁺ medicated crosslinking, showing significant, if not complete, burstrelease over the first 1-3 days.

FIG. 14a is a graph showing cumulative release (in micrograms) offluorescein conjugated insulin from hydrogels produced usingclick-conjugated LF 20/40 alginate at the concentration of 5% w/v thatwas oxidized to 5% or 10% and then reductively processed with AB.

FIG. 14b is a graph showing cumulative release (in micrograms) offluorescein conjugated insulin from hydrogels produced usingclick-conjugated LF 20/40 alginate at the concentration of 5% w/v thatwas oxidized to 5% or 10% and then oxidatively processed with SC.

FIG. 14c is a graph showing cumulative release (in micrograms) offluorescein conjugated IgG from hydrogels produced usingclick-conjugated LF 20/40 alginate at the concentration of 5% w/v thatwas oxidized to 5% or 10% and then reductively processed with AB.

FIG. 14d is a graph showing cumulative release (in micrograms) offluorescein conjugated IgG from hydrogels produced usingclick-conjugated LF 20/40 alginate at the concentration of 5% w/v thatwas oxidized to 5% or 10% and then oxidatively processed with SC.

FIG. 15a is a series of pictures of glass vials containing 4% MVGmaterial conjugated to Nb (left vial) and Tz (right vial) in thepresence of 2.3% gluteraldehyde, taken after 0 minutes, 40 minutes, 21.5hours and 67.5 hours, showing significant color change indicatingmodification of the click moieties.

FIG. 15b is a series of pictures of glass vials containing water ascontrol (left vial) or 2% MVG material conjugated to DBCO (middle vial)or azide (right vial), taken after 0 minutes, 40 minutes, 20 hours and69 hours, showing significant color change indicating modification ofthe click moieties.

FIG. 16a is a graph showing cumulative release (% load) of neutralliposomes (DOPC: Cholesterol) from Ca²⁺ cross-linked alginate and fromnon-oxidized click conjugated alginate over the period of 50 days inPBS⁺⁺ (a PBS buffer that contains Ca²⁺ and Mg²⁺ ions).

FIG. 16b is a graph showing cumulative release (% load) of neutralliposomes (DOPC: Cholesterol) from Ca²⁺ cross-linked alginate hydrogel,non-oxidized click conjugated alginate hydrogel and click gelatinhydrogel in PBS⁺⁺ (a PBS buffer that contains Ca²⁺ and Mg²⁺ ions).

FIG. 16c is a graph showing cumulative release (% load) of neutralliposomes (DOPC: Cholesterol) over the period of 28 days from Ca²⁺cross-linked alginate and from non-oxidized click conjugated alginatehydrogels prepared at 5% w/v alginate concentration. The liposomerelease profiles were measured in vitro in PBS⁻⁻, which is a PBS bufferthat does not contain Ca²⁺ or Mg²⁺ ions, and demonstrate thatclick-crosslinked gels are able to retain the encapsulated liposomes(diffusion limited), while liposomes are able to diffuse out of thecalcium crosslinked gels.

FIG. 16d is a graph showing cumulative release (% load) of neutralliposomes (DOPC: Cholesterol) from non-oxidized click conjugatedalginate hydrogels and click conjugated gelatin hydrogels over theperiod of 8 days. Gels were digested with alginate lyase or collagenase,respectively, on the 8^(th) day in order to show recovery and massbalance.

FIG. 17a is a dynamic light scattering (DLS) trace of the liposome stockmaterial that was encapsulated in the gels depicted in FIG. 17 d.

FIG. 17b is a DLS trace of a liposome which has been released from anon-oxidized click conjugated alginate hydrogel digested with alginatelyase after 8 days as depicted in FIG. 17d , showing minimal differencefrom the average diameter of the liposomes present in the stocksolution.

FIG. 17c is a DLS trace of a liposome which has been released after 28days from a non-oxidized click conjugated alginate hydrogel prepared atthe concentration of alginate of 5% w/v in PBS⁻ buffer, showing minimaldifference from the average diameter of the liposomes present in thestock solution.

FIG. 17d is a DLS trace of a liposome which has been released from aclick conjugated gelatin hydrogel digested with collagenase after 8days.

FIG. 17e is a DLS trace of a liposome which has been released after 3days from a calcium cross-linked alginate prepared in PBS⁻ buffer,showing that released liposomes are more polydisperse and of greaterdiameter than the liposome standard.

FIG. 17f is a DLS trace of a liposome which has been released after 28days from a calcium cross-linked hydrogel prepared at the alginateconcentration of 5% w/v in PBS⁻ buffer, showing that released liposomesare more polydisperse and of greater diameter than the liposomestandard.

FIG. 18 is a graph showing cumulative release (% load) of liposomes over75 days in PBS⁻ from click conjugated alginate hydrogels that wereoxidized to 0%, 5% and 10% total oxidation and reductively processedwith AB prior to click conjugation.

FIG. 19a is a picture of tubes containing alginate hydrogelsencapsulating liposomes and supernatants at day 0. To prepare alginatehydrogels, alginate was oxidized to 20% and then subsequently reducedwith ammonia borane. Gels were incubated at 37° C. in either sodiumcitrate buffer (pH 5; two vials on the left) or sodium borane (pH 9; twovials on the right).

FIG. 19b is a picture of tubes containing alginate hydrogelsencapsulating liposomes and supernatants at day 1. To prepare alginatehydrogels, alginate was oxidized to 20% and then subsequently reducedwith ammonia borane. Gels were incubated at 37° C. in either sodiumcitrate buffer (pH 5; two vials on the left) or sodium borane (pH 9; twovials on the right).

FIG. 19c is a picture of tubes containing alginate hydrogelsencapsulating liposomes and supernatants at day 7. To prepare alginatehydrogels, alginate was oxidized to 20% and then subsequently reducedwith ammonia borane. Gels were incubated at 37° C. in either sodiumcitrate buffer (pH 5; two vials on the left) or sodium borane (pH 9; twovials on the right). The pictures indicate that the pH 9 samples weredegraded after 7 days, while the pH 5 samples were still somewhatintact.

FIG. 19d is a picture of tubes alginate hydrogels encapsulatingliposomes and supernatants at day 14. To prepare alginate hydrogels,alginate was oxidized to 20% and then subsequently reduced with ammoniaborane. Gels were incubated at 37° C. in either sodium citrate buffer(pH 5; two vials on the left) or sodium borane (pH 9; two vials on theright). Images indicate that the samples were degraded after 14 daysboth at the pH 5 and pH 9.

FIG. 19e is a graph showing the degradation based release of neutralliposomes (DOPC: Cholesterol) from alginate hydrogels that have beenproduced by oxidizing alginate to 20% and subsequently reducing it withammonia borane. Samples were released in MES buffer pH 6.5 to mimic theparatumoral microenvironment.

FIG. 20a is a DLS trace of a DOTAP: Hydro Soy PC liposome prior toextrusion.

FIG. 20b is a DLS trace of DOTAP: Hydro Soy PC liposomes afterextrusion.

FIG. 20c is a graph showing cumulative release (% load) of cationicliposomes (FIG. 20a ) over the period of 17 days from Ca²⁺ cross-linkedalginate and from non-oxidized click conjugated alginate hydrogels. FIG.21b shows that click-crosslinked gels are able to retain theencapsulated liposomes (diffusion limited), while liposomes are able todiffuse out of the calcium crosslinked gels.

FIG. 21a is a graph showing the release of IGF-1 from click conjugatedalginate hydrogels prepared using various alginate compositions.

FIG. 21b is a graph showing the release of VEGF₁₆₅ from click alginatehydrogels prepared using various alginate compositions.

FIG. 22 is a bar graph showing the relative viscosities of variousalginate solutions at different alginate concentrations (% w/v), degreesof oxidation, and processing with either sodium chlorite or ammoniaborane. Data shows that click conjugation to MVG without oxidation doesnot result in substantial difference in viscosity, whereas the viscosityis substantially reduced with click conjugation to alginate containingeither algoxinol or algoxalate, and is comparable to that of water(0.890 cPa at 25° C.).

DETAILED DESCRIPTION OF THE INVENTION I. Reduced and Highly OxidizedPolysaccharides of the Invention

The present invention is directed to compositions comprising a reducedpolysaccharide, wherein the reduced polysaccharide comprises less than2% of residual aldehydes. The present invention is also directed tocompositions comprising a reduced polysaccharide, wherein the reducedpolysaccharide comprises less than 3% of residual aldehydes, and whereinthe polysaccharide is produced by reacting a polysaccharide with a diolspecific oxidizing agent, thereby producing an oxidized polysaccharidecomprising 15% or more oxidation on a molar basis, followed by reactingthe oxidized polysaccharide with a water soluble aldehyde specificreducing agent to produce the reduced polysaccharide.

The reduced polysaccharides comprised in the compositions of theinvention may be used for preparing hydrogels. These polysaccharides aregenerated by a two-step process. The first step involves reacting apolysaccharide, e.g., an alginate, with a diol specific oxidizing agentto produce an aldehyde containing oxidized polysaccharide, e.g., analdehyde containing oxidized alginate. The second step involves furtherreductive processing of the aldehyde containing oxidized polysaccharideto produce a reduced polysaccharide. The reduction process involvesreducing the aldehyde moieties present in the oxidized polysaccharide toproduce alcohol moieties. Accordingly, the term “reducedpolysaccharides”, as used herein, includes polysaccharides that comprisealcohol moieties. In some embodiments, the reduced polysaccharide isproduced by oxidizing the polysaccharide using a diol specific oxidizingagent to produce an aldehyde containing polysaccharide; and thenreductively processing the oxidized polysaccharide, e.g., by using awater soluble aldehyde specific reducing agent, to produce the reducedpolysaccharide. In certain examples, a reduced polysaccharide comprisesa monomeric subunit having a ring opened alcohol having the followingstructure:

The term “polysaccharide”, as used in this specification, refers to anypolymeric carbohydrate molecule composed of chains of monosaccharideunits bound together by glycosidic linkages. For example, alginate is apolysaccharide that comprises two different monomeric subunits,β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G), which are(1-4)-linked.

The polysaccharide chains may be linear or branched. The term“polysaccharide” is also intended to encompass an oligosaccharide. Thepolysaccharide can be a homopolysaccharide containing only monomer unitsof one kind (e.g., glucose), or a heteropolysaccharide, containingmonomer units of different kinds (e.g., glucose and fructose). In oneembodiment, the term “polysaccharide” refers to polymeric carbohydratemolecule that contains a diol, i.e., two hydroxyl groups present onadjacent carbons. Non-limiting examples of diol-containingpolysaccharides include alginate, pullulan, scleroglucan, chitosan,elsinan, xanthan gum, dextran, mannose, gellan, levan, cellulose,hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C,chondroitin sulfate E and β-d-glucans.

In one specific embodiment, the diol-containing polysaccharide is analginate polymer. Alginate polymers are comprised of two differentmonomeric units, (1-4)-linked (3-D-mannuronic acid (M units) and aL-guluronic acid (G units) monomers, which can vary in proportion andsequential distribution along the polymer chain. Alginate polymers arepolyelectrolyte systems which have a strong affinity for divalentcations (e.g., Ca⁺², Mg⁺², Ba⁺²) and form stable hydrogels when exposedto these molecules. See Martinsen A., et al., Biotech. & Bioeng., 33(1989) 79-89). For example, calcium cross-linked alginate hydrogels areuseful for dental applications, wound dressings chondrocytetransplantation and as a matrix for other cell types. Without wishing tobe bound by theory, it is believed that G units are preferentiallycrosslinked using calcium crosslinking, whereas click reaction basedcrosslinking is more indiscriminate with respect to G units or M units(i.e., both G and M units can be crosslinked by click chemistry).

The term “alginate”, used interchangeably with the term “alginatepolymers”, includes unmodified alginate, oxidized alginate (e.g.,comprising one or more algoxalate monomer units) and/or reduced alginate(e.g., comprising one or more algoxinol monomer units). In someembodiments, oxidized alginate comprises alginate comprising one or morealdehyde groups, or alginate comprising one or more carboxylate groups.In other embodiments, oxidized alginate comprises highly oxidizedalginate, e.g., comprising one or more algoxalate units. Oxidizedalginate may also comprise a relatively small number of aldehyde groups(e.g., less than 15%, e.g., 14,%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% 0.1%or less aldehyde groups or oxidation on a molar basis). The term“alginate” or “alginate polymers” may also include alginate, e.g.,unmodified alginate, oxidized alginate or reduced alginate, conjugatedto at least one click reagent as described below.

The alginate polymers useful in the context of the present invention mayhave an average molecular weight from about 20 kDa to about 500 kDa,e.g., from about 20 kDa to about 40 kDa, from about 30 kDa to about 70kDa, from about 50 kDa to about 150 kDa, from about 130 kDa to about 300kDa, from about 230 kDa to about 400 kDa, from about 300 kDa to about450 kDa, or from about 320 kDa to about 500 kDa. In one example, thealginate polymers useful in the present invention may have an averagemolecular weight of about 32 kDa. In another example, the alginatepolymers useful in the present invention may have an average molecularweight of about 265 kDa. In some embodiments, the alginate polymer has amolecular weight of less than about 1000 kDa, e.g., less than about 900Kda, less than about 800 kDa, less than about 700 kDa, less than about600 kDa, less than about 500 kDa, less than about 400 kDa, less thanabout 300 kDa, less than about 200 kDa, less than about 100 kDa, lessthan about 50 kDa, less than about 40 kDa, less than about 30 kDa orless than about 25 kDa. In some embodiments, the alginate polymer has amolecular weight of about 1000 kDa, e.g., about 900 Kda, about 800 kDa,about 700 kDa, about 600 kDa, about 500 kDa, about 400 kDa, about 300kDa, about 200 kDa, about 100 kDa, about 50 kDa, about 40 kDa, about 30kDa or about 25 kDa. In one embodiment, the molecular weight of thealginate polymers is about 20 kDa.

“GGGGRGDSP” disclosed as SEQ ID NO: 12.

A polysaccharide, e.g., a diol containing polysaccharide, such as analginate, may be reacted in the first step with a diol specificoxidizing agent. The term “diol specific oxidizing agent” refers to anoxidizing agent that specifically reacts with a diol moiety, e.g., adiol moiety present in a polysaccharide, and does not oxidize otherfunctional groups, e.g., alcohols, that may also be present in apolysaccharide. Non-limiting examples of diol specific oxidizing agentsinclude sodium periodate (NaIO₄), periodic acid (HIO₄), leadtetraacetate (PB(OAc)₄), sodium paraperiodate (Na₃H₂IO₆) and potassiumperiodate (KIO₄). In a specific embodiment, the diol specific oxidizingagent is sodium periodate (NaIO4).

The diol-specific oxidizing reagent reacts with a diol to cleave thecarbon-carbon bond and to produce two aldehyde moieties. In certainembodiments, this reaction produces oxidized polysaccharides that are atleast about 0.1%, 0.5%, 1%, about 5%, about 10%, about 15%, about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 95%, about 99% or about 100% oxidized. Rangesintermediate to the recited values are also intended to be part of thisinvention. For example, in certain embodiments, this reaction mayproduce oxidized polysaccharides that are about 1% to about 5%, about 3%to about 10%, about 5% to about 20%, about 10% to about 15%, about 12%to about 25%, about 15% to about 30%, about 20% to about 25%, about 25%to about 45%, about 30% to about 50%, about 45% to about 60%, about 50%to about 70%, about 55% to about 75%, about 60% to about 80%, about 65%to about 80%, about 70% to about 90% or about 85% to about 100%oxidized.

The term “oxidized polysaccharide”, e.g., “oxidized alginate”, as usedthroughout this specification, refers to a polysaccharide that has beenoxidized, e.g., reacted with an oxidizing agent, such as a diol specificoxidizing agent. An oxidized polysaccharide, e.g., an alginate,comprises aldehyde moieties which result from oxidation of thepolysaccharide. An oxidized polysaccharide may be about 0.1% to about100% oxidized, e.g., about 0.1% to about 5%, about 1% to about 10%,about 5% to about 20%, about 15% to about 40%, about 25% to about 60%,about 40% to about 70%, about 55% to about 90% or about 75% to about100% oxidized. In some embodiments, the oxidized polysaccharide may beless than about 15% oxidized, e.g., less than about 14%, less than about13%, less than about 12%, less than about 11%, less than about 10%, lessthan about 9%, less than about 8%, less than about 7%, less than about6%, less than about 5%, less than about 4%, less than about 3%, lessthan about 2%, less than about 1%, less than about 0.9%, less than about0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%,less than about 0.4%, less than about 0.3%, less than about 0.2%, orless than about 0.1% oxidized.

The term “% oxidation” or “% oxidized”, “oxidation level” or “%oxidation level”, used interchangeably with the term “% oxidation on amolar basis”, refers to % molar fraction of monomeric subunits in a diolcontaining polysaccharide, e.g., an alginate, that are ring opened as aresult of oxidation. In some cases, the polysaccharide is a diolcontaining polysaccharide that has been subjected to a reaction with adiol specific oxidizing agent, such as sodium periodate. Methods thatmay be useful for measuring % oxidation of a polysaccharide are known toone of ordinary skill in the art and may include, e.g., qNMR.

For example, the term “% oxidation” may be used in reference to apolysaccharide that has been oxidized, e.g., by a diol specificoxidizing agent, and contains ring opened monomeric subunits comprisingaldehyde moieties. One example of such monomeric subunit, e.g., inalginate, is shown below:

The term “% oxidation” may also be used in reference to a polysaccharidethat has been oxidized, e.g., by a diol specific oxidizing agent, andhas subsequently been reduced, e.g., by a water soluble aldehydespecific reducing agent, and contains ring opened monomeric subunitscomprising alcohol moieties. One example of such monomeric subunit,e.g., algoxinol, is shown below:

The term “% oxidation” may also be used in reference to a polysaccharidethat has been oxidized, e.g., by a diol specific oxidizing agent, andhas subsequently been further oxidized, e.g., by a second oxidizingagent, and contains ring opened monomeric subunits comprising carboxylicacid moieties. One example of such monomeric subunit, e.g., algoxalate,is shown below:

The term “% aldehydes”, as used herein, refers to the % molar fractionof aldehydes present in ring opened monomeric subunits of apolysaccharide, e.g., a diol containing polysaccharide, e.g., analginate.

The term “% residual oxidation”, “residual oxidation level”, “% residualoxidation level”, “% residual oxidation on a molar basis” or “% residualaldehydes” refers to the % molar fraction of residual aldehydes, i.e.,aldehyde moieties that remain in a polysaccharide after it has beenreacted with a diol specific oxidizing agent and then reduced, e.g.,with an aldehyde specific water soluble reducing agent, to producereduced polysaccharide as described below. In some embodiments, thereduced polysaccharide, e.g., the reduced alginate, may comprise about0.01% to about 3% residual aldehydes, e.g., about 0.01% to about 1%,about 0.05 to about 1.5% or about 1% to about 3% residual aldehydes. Forexample, the reduced polysaccharide may comprise about 2% residualaldehydes or about 3% residual aldehydes. This term may also refer tothe % molar fraction of residual aldehydes, i.e., aldehyde moieties thatremain in a polysaccharide after it has been reacted with a diolspecific oxidizing agent and then further oxidized, e.g., with a secondoxidizing agent, to produce highly oxidized polysaccharide as describedbelow. In some embodiments, the highly oxidized polysaccharide, e.g.,the highly oxidized alginate, may comprise about 0.01% to about 3%residual aldehydes, e.g., about 0.01% to about 1%, about 0.05 to about1.5% or about 1% to about 3% residual aldehydes. For example, the highlyoxidized polysaccharide may comprise about 2% residual aldehydes orabout 3% residual aldehydes.

An exemplary reaction between a monomeric unit of a polysaccharide isthe reaction between alginate and sodium periodate, a diol specificoxidizing agent, is illustrated in Scheme 1:

Further reductive processing of the aldehyde containing oxidizedpolysaccharides in the second step of the methods of the inventioninvolves a reaction of the aldehyde containing oxidized polysaccharidewith an aldehyde specific water soluble reducing agent. The term“aldehyde specific water soluble reducing agent” refers to a reducingagent that specifically oxidizes aldehydes, e.g., aldehydes present in apolysaccharide as a result of oxidation by a diol specific oxidizingagent, and converts them to alcohols. In certain embodiments, thealdehyde specific water soluble reagent is non-toxic and/or is a Greenreagent. Non-limiting examples of aldehyde specific water solublereducing agents include sodium borohydride (NaBH₄); sodiumcyanoborohydride (NaCNBH₃); hydrogen gas (H₂) in the presence of acatalyst, e.g., a nickel (Ni), a platinum (Pl) or a palladium (Pd)catalyst; ammonia borane (H₃NBH₃); or a borane complex, e.g., abis-carbonate borane complex ([(BH₃)₂CO₂]²⁻. 2Na⁺), a boranedimethylamine complex [(CH₃)₂NH.BH₃]; a borane tert-butylamine complex[(CH₃)₃CNH₂.BH₃]; or a borane-pyrimidine complex. In a specific example,the aldehyde specific water soluble reducing agent is ammonia borane. Inone embodiment, the aldehyde specific water soluble reducing agent isnot sodium borohydride.

An exemplary reaction of the oxidized glucuronic acid, a product of thereaction illustrated in Scheme 1, with ammonia borane, a water solublealdehyde specific reducing agent, is illustrated in Scheme 2.

In the context of the present invention, the term “residual aldehyde”refers to the aldehydes remaining in a reduced polysaccharide after thereduction of the oxidized polysaccharide, e.g., oxidized alginate, withan aldehyde specific water soluble reducing agent, e.g., ammonia borane,is completed. The amount of aldehydes present in a polysaccharide, e.g.,the amount of residual aldehydes present in a polysaccharide afterreduction with a water soluble aldehyde specific reducing agent, may bemeasured by any methods known to one of skill in the art. For example,the amount of residual aldehydes present in a polysaccharide may bemeasured by quantitative NMR, where the integral of alginate (1.5% w/v)aldehyde peaks (>5.1 ppm) are compared to an internal standarddimethylmalonic acid (2.5 mg/mL), and may be expressed as % of residualaldehydes.

It has been surprisingly discovered by the present inventors that watersoluble aldehyde specific reducing agents, e.g., ammonia borane, areparticularly effective at reducing aldehydes present in an oxidizedpolysaccharide, e.g., an alginate polymer, and converting them intoalcohols. Accordingly, a reduced polysaccharide that has been obtainedfrom subjecting an oxidized polysaccharide to further reductiveprocessing via a reaction with a water soluble aldehyde specificreducing agent, e.g., ammonia borane, contains surprisingly low levelsof residual aldehydes. In some embodiments, the reduced polysaccharidecontains less than 2% of residual aldehydes, e.g., less than 1.5%, lessthan 1% or less than 0.5% of residual aldehydes. In other embodiments,the reduced polysaccharide contains less than 3% of residual aldehydes,e.g., less than 3%, less than 2.5%, less than 2%, less than 1.5%, lessthan 1%, or less than 0.5% of residual aldehydes.

It is desirable to have a low level of aldehydes in a polysaccharide,e.g., an alginate, used to prepare a hydrogel, because aldehydes cancause damage to proteins and other molecules present in the vicinity ofthe polysaccharide, thereby increasing the toxicity of the hydrogel.Additionally, the use of water soluble aldehyde specific reducing agentsis particularly advantageous because these reagents do not produce toxicby-products and do not necessitate further processing of thepolysaccharide materials. The water soluble aldehyde specific reducingagents, e.g., ammonia borane, are also non-toxic and/or are consideredto be “Green Reagents”.

The present invention also utilizes highly oxidized polysaccharides,e.g., highly oxidized alginate polymers. These highly charged oxidizedpolysaccharides are more soluble in water than oxidized polysaccharides,making their manipulation simpler. The highly oxidized polysaccharidesmay be prepared in two steps. The first step involves reacting apolysaccharide, e.g., an alginate polymer, with a diol specificoxidizing agent to produce an aldehyde containing oxidizedpolysaccharide, e.g., an aldehyde containing oxidized alginate polymer,as described above. The second step involves further oxidativeprocessing of the aldehyde containing oxidized polysaccharide to producea highly oxidized polysaccharide. The further oxidative processing ofthe oxidized polysaccharide involves converting the aldehyde moietiespresent in the oxidized polysaccharide into carboxylic acid moieties.Accordingly, the term “highly oxidized polysaccharide”, as used herein,includes polysaccharides that comprise at least one, e.g., twoadditional carboxylic acid moieties per monomeric subunit. For example,a highly oxidized polysaccharide may comprise a monomeric subunit whichis a ring opened carboxylic acid containing alginate, or algoxalate,having the following structure:

The further oxidative processing of the aldehyde containing oxidizedpolysaccharide, e.g., an alginate polymer, involves a reaction of thealdehyde containing oxidized polysaccharide with a second oxidizingagent. The term “second oxidizing agent”, as used in this specification,refers to any oxidizing agent that specifically converts an aldehydemoiety into a carboxylic acid moiety, thereby producing a highlyoxidized polysaccharide. Non-limiting examples of the second oxidizingagents include sodium chlorite; bromine; dilute nitric acid (NHO₃);silver oxide, e.g., Tollens' Reagent ([Ag(NH₃)₂]⁺ or AgNO₃); copper(II)complexes, e.g., Fehling's reagent (copper (II) tartrate solution), orBenedict's reagent (copper (II) citrate solution); potassiumpermanganate (KMnO₄); and hydrogen peroxide (H₂O₂). In a specificembodiment, the second oxidizing agent is sodium chlorite.

An exemplary reaction of the oxidized monomeric unit of a polysaccharideis the reaction of oxidized alginate, a product of the reactionillustrated in Scheme 1, with sodium chlorite, a second oxidizing agent,is illustrated in Scheme 3.

It has been surprisingly discovered that both reduced polysaccharides,e.g., reduced polysaccharides resulting from the reaction of oxidizedpolysaccharides with water soluble aldehyde specific reducing agents(e.g., ammonia borane), such as polysaccharides comprising algoxinol, aswell as highly oxidized polysaccharides, e.g., the polysaccharidesresulting from the reaction of oxidized polysaccharides with a secondoxidizing agent, such as polysaccharides comprising algoxalate, areparticularly useful for preparing hydrogels. The hydrogels prepared fromreduced and highly oxidized polysaccharides, e.g., reduced and highlyoxidized alginate polymers, are biodegradable and useful as drugdelivery vehicles.

In some embodiments, the solubility of reduced and/or highly oxidizedpolysaccharide, e.g., reduced and/or highly oxidized alginate, such asalginate comprising algoxinol and/or algoxalate, is higher than thesolubility of unmodified alginate. The solubility of a polysaccharide,such as a reduced and/or a highly oxidized polysaccharide, may beexpressed as % w/v or in mg/mL, wherein 1% w/v is equivalent to 10 mg/mLof the polysaccharide. For example, the solubility of reduced and/orhighly oxidized polysaccharide may be about 10-20% w/v, 15-30% w/v,20-40% w/v, 30-50% w/v/, 40-65% w/v, 50-75% w/v or 60-90% w/v. Forexample, the solubility of the reduced and/or highly oxidizedpolysaccharide of the invention may be about 10% w/v, about 15% w/v,about 20%, about 25% w/v, about 30% w/v, about 35% w/v, about 40% w/v,about 45% w/v, about 50% w/v, about 55% w/v, about 60% w/v, about 65%w/v, about 70% w/v, about 75% w/v, about 80% w/v or about 90% w/v.

In some embodiments, the polysaccharides of the invention arebiodegradable. For example, oxidized, reduced and highly oxidizedalginate is biodegradable. Alginate, e.g., oxidized, reduced and highlyoxidized alginate, is not susceptible to degradation by a hostendogenous enzyme, e.g., an endogenous enzyme that may be present in ahuman. Alginate, e.g., oxidized, reduced and highly oxidized alginate,may be chemically degraded, e.g., hydrolyzed when exposed to acidic oralkaline condition. Acidic conditions comprising a pH of 6.5 or lower,while alkaline conditions comprise a pH of 8 or higher.

II. Polysaccharides of the Invention Conjugated to Click Reagents

The reduced and highly oxidized polysaccharides of the invention, e.g.,alginate, may be conjugated with a click reagent. The term “clickreagent”, used in this specification interchangeably with the term“click chemistry reagent” is a reagent that can rapidly and selectivelyreact (“click”) with its counterpart click reagent under mild conditionsin aqueous solution. The mild conditions include neutral pH, aqueoussolution and ambient temperature, with low reactant concentrations.Exemplary click pair reagents are well known to one of skill in the artand include, but are not limited to, azide and dibenzocyclooctyne(DBCO), tetrazine and transcyclooctene, and tetrazine and norbornene,with the structures illustrated below.

In some embodiments, the click reagent is tetrazine (Tz). As usedherein, the terms “tetrazine” and “tetrazine moiety” include moleculesthat comprise 1,2,4,5-tetrazine substituted with suitable spacer forlinking to the polymer (e.g., alkylamines like methylamine orpentylamine), and optionally further substituted with one or moresubstituents at any available position. Exemplary tetrazine moietiessuitable for the compositions and methods of the disclosure include, butare not limited to, the structures shown below (see, e.g., Karver etal., (2011) Bioconjugate Chem. 22:2263-2270, and WO 2014/065860, theentire contents of each of which are hereby incorporated herein byreference):

One of the counterpart reagents for tetrazine is norbornene (Nb). Asused herein, the terms “norbornene” and “norbornene moieties” includebut are not limited to norbornadiene and norbornene groups furthercomprising suitable spacer for linking to the polysaccharide (e.g.,alkylamines like methylamine or pentylamine), and optionally furthersubstituted with one or more substituents at any available position.Such moieties include, for example, norbornene-5-methylamine andnorbornadienemethylamine.

Click reagents are typically conjugated to a polysaccharide, e.g., analginate, via the carboxylic moiety. In one specific example illustratedin FIG. 1a , click reagents may be conjugated to alginate polymers viathe carboxylate moiety present in the glucuronic acid in an alginate.Accordingly, one click molecule may be conjugated per glucuronate in anunprocessed alginate. Following oxidation of alginate with sodiumperiodate, two aldehydes per glucuronate are generated. Each aldehydemoiety may become conjugated to a click reagent via an imine bond (FIG.1b ). This conjugation of click reagents to oxidized alginates issub-optimal because of residual aldehydes that remain in the alginatefollowing conjugation that may result in toxicity and damage to cargo,as well as in degradation of the click reagents (see also Examples 1 and3). Further, the imine bonds between the aldehydes and the clickreagents are easily hydrolyzable, resulting in the loss of clickreagents from the alginate. As shown in FIG. 1c , further oxidation ofaldehydes present in oxidized alginates converts these aldehydes intocarboxylic acids thereby providing two additional sites for clickconjugation.

It has been surprisingly discovered that certain highly oxidizedpolysaccharides, e.g., highly oxidized alginate polymers, are bettersubstrates for click reagent conjugation than oxidized polysaccharidesbecause they contain additional sites available for click conjugation.It has also been discovered that reduced polysaccharides, e.g., reducedalginates, are better suited for click reagent conjugation than oxidizedpolysaccharides because they contain a lower amount of aldehydes thatcan react with click reagents, causing their degradation.

In some embodiments, the click reagent conjugated to a polysaccharide ofthe invention, e.g., an alginate, may also react with its counterpartclick reagent that is, in turn, attached to a moiety, therebyconjugating the moiety to the polysaccharide. Any moiety may beconjugated to the polysaccharide of the invention using the clickreagents. Non-limiting examples of such moieties include a small organicmolecule, a small inorganic molecule; a saccharine; a monosaccharide; adisaccharide; a trisaccharide; an oligosaccharide; a polysaccharide; apeptide; a protein, a peptide analog, a peptide derivative; apeptidomimetic; an antibody (polyclonal or monoclonal); an antigenbinding fragment of an antibody; a nucleic acid, e.g., anoligonucleotide, an antisense oligonucleotide, siRNAs, shRNAs, aribozyme, an aptamer, microRNAs, pre-microRNAs, iRNAs, plasmid DNA (e.g.a condensed plasmid DNA), a modified RNA, and a nucleic acid analog orderivative. In some embodiments, the moiety is a therapeutic agent.

In other embodiments, a click reagent conjugated to a polysaccharide ofthe invention, e.g., an alginate, may function as a cross-linking agent,as described in detail below.

In certain embodiments, a polysaccharide of the invention, e.g., analginate, may be conjugated to two or more click reagents, that belongto different click pairs. For example, in an embodiment where thepolysaccharide of the invention is conjugated to two click reagentsbelonging to two different click pairs, a first click reagent conjugatedto the polysaccharide may be azide from the azide-dibenzocyclooctyne(azide-DBCO) click pair, and a second click reagent conjugated to thepolysaccahride may be tetrazine from the tetrazine-norbornene clickpair. In such polysaccharides, the two click reagents may be used fordifferent functions. For example, the first click reagent may functionto promote cross-linking of the polysaccharide to form a hydrogel, andthe second cross-linking reagent may function to conjugate a moiety tothe polysaccharide, as described above. In a specific embodiment, when apolysaccharide of the invention is conjugated to two or more clickreagents, the click reagents conjugated to the polysaccharide do notcross-react, i.e., do not react with each other, but only react withtheir click pairs.

III. Polysaccharides of the Invention Conjugated to Cross-Linking Agents

The reduced and highly oxidized polysaccharides of the invention may beconjugated with a cross-linking agent. The cross-linking agent may beattached to the oxidized polysaccharide ionically, covalently orphysically. In one embodiment, the cross-linking agent is covalently ornon-covalently attached to the reduced polysaccharide of the invention,e.g., a reduced alginate polymer. In another embodiment, thecross-linking agent is covalently or non-covalently attached to thehighly oxidized polysaccharide of the invention, e.g., a highly oxidizedalginate polymer.

The term “cross-linking agent”, as used in this specification, is anyagent that may effect and promote cross-linking of the polysaccharide toa specific location, e.g., a location inside a cell or a tissue of asubject, or that may promote cross-linking of a polysaccharide, e.g., analginate polymer. In certain embodiments, the cross-linking agent isattached to the polysaccharide, e.g., via a covalent bond. In certainembodiments, the cross-linking agent is only coupled to one polymerchain and functions as a pendant group.

In some embodiments, the cross-linking agent is a click reagent.Exemplary pairs of click reagents that may be used for cross-linking thepolysaccharide of the invention include, but are not limited to, azideand dibenzocyclooctyne (DBCO), tetrazine and trancyclooctene, andtetrazine and norbornene, with structures as illustrated above.

The reduced and/or highly oxidized polysaccharide of the inventionmodified with a click reagent, e.g., an alginate conjugated to a clickreagent, can be covalently cross-linked to form a click-crosslinkedhydrogel, e.g., a click alginate hydrogel. Formation of hydrogels viaclick chemistry is described in Desai et al. (2015) Biomaterials50:30-37, the entire contents of which are hereby incorporated herein byreference. The cross-linking reaction has been previously shown byothers to be highly specific, bio-orthogonal, and quick (see, e.g.,Devaraj et al. (2008), Bioconjugate Chem. 19(12):2297-2299; Karver etal. (2011) Bioconjugate Chem. 22(11):2263-2270; and Alge et al. (2013)Biomacromol. 14(4):949-953), allowing for incorporation of cells withhigh post-encapsulation viability.

For example, to generate a hydrogel from a click-conjugatedpolysaccharide of the invention, e.g., a click conjugated alginate,e.g., comprising algoxinol and/or algoxalate, a polysaccharideconjugated to one member of a click pair, e.g., Nb may be mixed with apolysaccharide conjugated to the second member of a click pair, e.g., Tzand incubated at mild conditions to generate the hydrogel. Properties ofsuch hydrogels, e.g., stiffness of the hydrogel, the time to gelation,or an average pore size of the hydrogel, may be modulated by varying anumber of parameters that include the degree of click conjugation of thepolysaccharide; the degree of polysaccharide oxidation, i.e., % residualaldehydes present in the polysaccharide; and the concentration of clickconjugated polysaccharide during hydrogel formation, e.g., % w/v/ of thealginate material present in the solution immediately prior to formationof the hydrogel.

The term “degree of click conjugation”, which may be usedinterchangeably with the term “degree of substitution” or “DS” refers tothe average number of click reagents per monomeric unit of apolysaccharide, e.g., the average number of click reagents per monomerunits in an alginate. The degree of click substitution may be varied byvarying the number of molar equivalents of click reagent to the moles ofalginate monomers in the click conjugation reaction. For example, theclick conjugation reaction may comprise about 1 to about 5000 molarequivalents of a click reagent, e.g., about 1, about 5, about 10, about20, about 50, about 100, about 150, about 200, about 250, about 300,about 350, about 400, about 450, about 500, about 550, about 600, about650, about 700, about 750, about 800, about 850, about 900, about 950,about 1000, about 1200, about 1500, about 1800, about 2000, about 2200,about 2500, about 2800, about 3000, about 3200, about 3500, about 4000,about 4200, about 4500 or about 5000 molar equivalents of the clickreagent. Ranges intermediate to the recited values are also intended tobe part of this invention. For example, the click conjugation reactionmay comprise about 1 to about 50, about 10 to about 100, about 150 toabout 250, about 200 to about 500, about 400 to about 800, about 700 toabout 1000, about 1200 to about 1600, about 1500 to about 2000, about1800 to about 3500, or about 3200 to about 5000 molar equivalents of aclick reagent.

In some embodiments, the polysaccharide of the invention conjugated to aclick reagent comprises a degree of click substitution that is about0.01% to about 100%, e.g., about 0.01% to about 0.5%, about 0.1% toabout 5%, about 1% to about 10%, about 5% to about 15%, about 10% toabout 20%, about 15% to about 25%, about 20% to about 35%, about 30% toabout 40%, about 35% to about 50%, about 40% to about 60%, about 50% toabout 75%, about 70% to about 90% or about 85% to about 100%. Forexample, the polysaccharide of the invention conjugated to a clickreagent may be about 0.01%, about 0.05%, about 0.1%, about 0.5%, about1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% orabout 100%.

In some embodiments, the concentration of click conjugatedpolysaccharide during hydrogel formation, e.g., concentration of thealginate material present in the solution immediately prior to formationof the hydrogel, may be from about 0.01% to about 50% w/v, e.g., about0.01% to about 10% w/v, about 0.1% to about 5% w/v, about 1% to about15% w/v, about 10% to about 30% w/v, about 12% to about 35% w/v, about15% to about 25% w/v, about 20% to about 45% w/v or about 35% to about50% w/v. For example, the concentration of the alginate material presentin the solution immediately prior to formation of the hydrogel may beabout 0.01%, about 0.05%, about 1%, about 0.5%, about 1%, about 2%,about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45% or about 50%.

Stiffness of the hydrogel may depend on the degree of click substitutionand may be determined by measuring an elastic modulus (or a storagemodulus) G′. For example, G′ of a hydrogel of the invention may varyfrom about 0 to about 40000 Pa when measured at 1% strain at 1 Hz, e.g.,about 0 to about 100 Pa, about 50 to about 300 Pa, about 100 to about450 Pa, about 130 to about 600 Pa, about 300 to about 450 Pa, about 500to about 900 Pa, about 600 to about 1500 Pa, about 1200 to about 2000Pa, or about 1900 to about 40000 Pa. For example, G′ of a hydrogel ofthe invention when measured at 1% strain at 1 Hz, may be about 100 Pa,about 200 Pa, about 500 Pa, about 1000 Pa, about 1500 Pa, about 2000 Pa,about 2200 Pa, about 2500 Pa or about 3000 Pa.

In some embodiments, the cross-linking agent is a peptide, e.g., a celladhesive peptide. Accordingly, a reduced and/or highly oxidizedpolysaccharide of the invention, e.g., a reduced and/or highly oxidizedalginate polymer, may be modified by a cell adhesive peptide, e.g., anextracellular cell matrix (ECM) component. The cell adhesive peptide cancomprise, for example, the amino acid sequencearginine-glycine-aspartate (RGD). Examples include the amino acidsequence arginine-glycine-aspartate-cysteine (RGDC) (SEQ ID NO: 1) andarginine-glycine-aspartate-serine (RGDS) (SEQ ID NO: 2). In otherexamples, the cell adhesive peptide comprises the amino acid sequence oflysine-glutamine-alanine-glycine-aspartate-valine (KQAGDV) (SEQ ID NO:3) or valine-alanine-proline-glycine (VAPG) (SEQ ID NO: 4). In someexamples, the cell adhesive peptide is CGGGGRGDSP (SEQ ID NO: 5). Othercell adhesive peptides may be used based on the desired application andwill be apparent to one of skill in the art.

In some cases, the cell adhesive peptide is covalently linked to thereduced and/or highly oxidized polysaccharide via a thiol-ene reaction,e.g., via thiol-ene photochemistry. For example, the cell adhesivepeptide can be covalently linked to the polysaccharide (e.g., analginate polymer) prior to or following crosslinking of thepolysaccharide to form a hydrogel. Such use of thiol-ene reaction tocovalently link the cell adhesive peptide to the polysaccharide issignificantly faster and more efficient than the previously disclosedmethods, such as methods using of carboxyl activating agents (e.g., EDC)to couple the peptide to the polymer.

IV. Hydrogels of the Invention

The reduced and/or highly oxidized polysaccharides described herein maybe used to prepare hydrogels for therapeutic applications. The hydrogelsprepared from the reduced and/or highly oxidized polysaccharides, e.g.,reduced and/or highly oxidized alginates, are biodegradable andnon-toxic, as compared to the hydrogels prepared from the oxidizedpolysaccharides, e.g., oxidized alginates. In some embodiments, when thehydrogels prepared using the reduced and/or highly oxidizedpolysaccharides of the invention encapsulate a cell, less cell toxicityis observed, as compared to the hydrogel prepared from the oxidizedpolysaccharides. In other embodiments, in which the hydrogels producedfrom the reduced and/or highly oxidized polysaccharides of the inventionare used to encapsulate a therapeutic agent, e.g., a protein, lessprotein damage is observed, as compared to hydrogel prepared fromoxidized polysaccharides. In embodiments, in which the hydrogelsproduced from the reduced and/or highly oxidized polysaccharides of theinvention are used to encapsulate a lipid based nanoparticle, e.g., aliposome or a virosome, the lipid based nanoparticle is delivered intactto a desired location within a host.

The hydrogels of the invention may be used to prepare a dosage formcomprising a therapeutic or a diagnostic agent encapsulated by thehydrogels of the invention. The hydrogels of the invention may also beused to prepare an implantable device which may comprise a therapeuticor a diagnostic agent. The hydrogels of the invention may also be usedto prepare a drug delivery composition comprising a lipid basednanoparticle, e.g., a liposome or a virosome encapsulating a therapeuticor a diagnostic agent and the hydrogel of the invention encapsulatingthe lipid based nanoparticle.

It has been presently discovered that the reduced and/or highly oxidizedpolysaccharides of the invention are useful for preparing hydrogels inwhich an average mesh size, measured as an average diameter of thepores, varies over a relatively wide range. For example, the hydrogelsprepared using the reduced and/or highly oxidized polysaccharides of theinvention may have pores with an average diameter ranging from about 0.5nm to about 500 nm, e.g., about 0.5 nm to about 1 nm, about 0.5 nm toabout 5 nm, about 1 nm to about 20 nm, about 10 nm to about 50 nm, about25 nm to about 80 nm, about 50 nm to about 100 nm, about 70 nm to about150 nm, about 100 nm to about 250 nm, about 200 nm to about 350 nm,about 250 nm to about 400 nm or about 350 nm to about 500 nm. In someembodiments, the hydrogels of the invention may have pores with anaverage diameter of about 0.5 nm, about 1 nm, about 5 nm, about 10 nm,about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm,about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about95 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300nm, about 350 nm, about 400 nm, about 450 nm or about 500 nm. Rangesintermediate to the recited values are also intended to be part of thisinvention.

In one example, the hydrogels of the invention have a surprisingly smallaverage pore size, e.g., an average diameter of about 10 nm or less. Insome embodiments, the diameter of the pore in a hydrogel of theinvention is about 10.00 nm or less, about 9.5 nm or less, about 9.0 nmor less, about 8.5 nm or less, about 8.0 nm or less, about 7.5 nm orless, about 7.0 nm or less, about 6.5 nm or less, about 6.0 nm or less,about 5.5 nm or less, about 5.0 nm or less, about 4.5 nm or less, about4.0 nm or less, about 3.5 nm or less, about 3.0 nm or less, about 2.5 nmor less, about 2.0 nm or less, about 1.5 nm or less or about 1 nm orless. For example, the hydrogel of the invention may comprise poreshaving an average diameter of about 10 nm to about 1 nm, about 10 nm toabout 4 nm, about 7 nm to about 3 nm, about 5 nm to about 1 nm, about 4nm to about 2 nm, or about 3 nm to about 0.5 nm. Ranges intermediate tothe recited values are also intended to be part of this invention.

Accordingly, the hydrogels of the invention may be used to encapsulate atherapeutic or diagnostic agent, or to encapsulate a lipid basednanoparticle, e.g., a liposome or a virosome comprising a therapeutic ordiagnostic agent, of a small molecular weight. For example, thehydrogels of the invention may be used to encapsulate therapeutic ordiagnostic agents, or a lipid based nanoparticle, e.g., a liposome or avirosome, encapsulating therapeutic or diagnostic agents, e.g.,polypeptides, having a molecular weight of 1000 kDa or less, e.g., 950kDa or less, 900 kDa or less, 850 kDa or less, 800 kDa or less, 750 kDaor less, 700 kDa or less, 650 kDa or less, 600 kDa or less, 550 kDa orless, 500 kDa or less, 450 kDa or less, 400 kDa or less, 350 kDa orless, 300 kDa or less, 250 kDa or less, 200 kDa or less, 150 kDa orless, 100 kDa or less, 90 kDa or less, 80 kDa or less, 70 kDa or less,60 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, 20 kDaor less, 10 kDa or less, 8 kDa or less, 6 kDa or less, 4 kDa or less, 2kDa or less, 1 kDa or less, 0.8 kDa or less, 0.6 kDa or less, 0.4 kDa orless, 0.2 kDa or less, or 0.1 kDa or less.

In some embodiments, a therapeutic or diagnostic agent that may beencapsulated by the hydrogel of the invention, or by a lipid basednanoparticle, e.g., a liposome or a virosome, encapsulated by thehydrogel of the invention, does not diffuse through the pores of thehydrogel because the pores of the hydrogel are sufficiently small ascompared to the size of the therapeutic agent. Accordingly, the releaseof the therapeutic agent from the hydrogel occurs gradually uponbiodegradation of the hydrogel inside a subject. This provides sustainedrelease of the therapeutic or diagnostic agent, or a lipid basednanoparticle, e.g., a liposome or a virosome, encapsulating thetherapeutic or diagnostic agent, in the subject, e.g., a human.

As used herein, the term “therapeutic or diagnostic agent” that isencapsulated by the hydrogels of the invention, includes any agent thatmay be used to treat, prevent or diagnose a disorder in a subject inneed thereof. A therapeutic or diagnostic agent may be a cell, e.g., amammalian cell, such as a human mesenchymal stem cell (hMSC), a smallmolecule or a biologic. The biologic may be a peptide, a protein, a DNAmolecule, an RNA molecule, a PNA molecule, an antibody or a vaccine.Exemplary therapeutic agents include, but are not limited to, thosefound in Harrison's Principles of Internal Medicine, 13^(th) Edition,Eds. T. R. Harrison et al. McGraw-Hill N.Y., NY; Physicians' DeskReference, 50^(th) Edition, 1997, Oradell N.J., Medical Economics Co.;Pharmacological Basis of Therapeutics, 8^(th) Edition, Goodman andGilman, 1990; United States Pharmacopeia, The National Formulary, USPXII NF XVII, 1990; current edition of Goodman and Oilman's ThePharmacological Basis of Therapeutics; and current edition of The MerckIndex, the entire contents of all of which are incorporated herein byreference.

In some embodiments, a therapeutic or diagnostic agent encapsulated bythe hydrogel of the invention, or by a lipid based nanoparticle, e.g., aliposome or a virosome, encapsulated by the hydrogel of the invention,may comprise a compound selected from the group consisting of a smallorganic molecule, a small inorganic molecule; a saccharine; amonosaccharide; a disaccharide; a trisaccharide; an oligosaccharide; apolysaccharide; a peptide; a protein; a peptide analog; a peptidederivative; a peptidomimetic; an antibody (polyclonal or monoclonal); anantigen binding fragment of an antibody; a nucleic acid, e.g., anoligonucleotide, an antisense oligonucleotide, siRNAs, shRNAs, aribozyme, an aptamer, microRNAs, a pre-microRNAs, iRNAs, plasmid DNA(e.g. a condensed plasmid DNA), modified RNA, a nucleic acid analog orderivative; an extract made from biological materials such as bacteria,plants, fungi, or animal cells; animal tissues; naturally occurring orsynthetic compositions; and any combinations thereof. The nucleic acidmay comprise one or more unnatural nucleotides. The peptide or theprotein may comprise one or more unnatural amino acids.

As used herein, the term “small molecule” can refer to a compound thatis “natural product-like,” however, the term “small molecule” is notlimited to “natural product-like” compounds. Rather, a small molecule istypically characterized in that it contains several carbon-carbon bonds,and has a molecular weight of less than 5000 Daltons (5 kD), preferablyless than 3 kD, still more preferably less than 2 kD, and mostpreferably less than 1 kD. In some cases, it is preferred that a smallmolecule have a molecular mass equal to or less than 700 Daltons.

As used herein, the term “peptide” is used in its broadest sense torefer to compounds containing amino acids, amino acid equivalents orother non-amino groups, while still retaining the desired functionalactivity of a peptide. Peptide equivalents can differ from conventionalpeptides by the replacement of one or more amino acids with relatedorganic acids (such as PABA), amino acids or the like or thesubstitution or modification of side chains or functional groups. Thepeptides can be linear or cyclic. A peptide can be modified to includeone or more of D-amino acids, beta-amino acids, chemically modifiedamino acids, naturally occurring non-proteogenic amino acids, rare aminoacids, and chemically synthesized compounds that have properties knownin the art to be characteristic of an amino acid.

As used herein, the term “nucleic acid” or “oligonucleotide” means atleast two nucleotides, including analogs or derivatives thereof, thatare covalently linked together. Exemplary oligonucleotides include, butare not limited to, single-stranded and double-stranded siRNAs and otherRNA interference reagents (RNAi agents or iRNA agents), shRNA (shorthairpin RNAs), antisense oligonucleotides, aptamers, ribozymes, andmicroRNAs (miRNAs). The nucleic acids can be single stranded or doublestranded. The nucleic acid can be DNA, RNA or a hybrid, where thenucleic acid contains any combination of deoxyribo- andribo-nucleotides, and any combination of uracil, adenine, thymine,cytosine and guanine. An RNA molecule may be selected from the groupconsisting of an mRNA, an RNAi, an siRNA, an shRNA, a microRNA, anisRNA, a lncRNA and an antisense RNA.

The nucleic acid may also include one or more unnatural nucleotides. Forexample, the nucleic acid can comprise one or more nucleic acidmodifications known in the art. For example, the nucleic acids cancomprise one or more backbone modifications, e.g., phosphoramide(Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein;Letsinger, J. Org. Chem. 35:3800 (1970)), phosphorothioate,phosphorodithioate, O-methylphophoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), or peptide nucleic acid linkages (see Egholm, J. Am. Chem. Soc.114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); andNielsen, Nature, 365:566 (1993), the entire contents of all of which areherein incorporated by reference. The nucleic acids can also includemodifications to nucleobase and/or sugar moieties of nucleotides.Exemplary sugar modifications at the sugar moiety include replacement of2′-OH with halogens (e.g., fluoro), O-mehtyl, O-methoxyethyl, NH₂, SHand S-methyl.

In some embodiments, the term “therapeutic or diagnostic agent”comprises biological material, for example, an extracellular matrixmaterial such as fibronectin, vitronection and laminin; a cytokines; agrowth factor; a differentiation factor, a nucleic acid; a protein; apeptide; an antibody or a fragment thereof or an antigen binding portionthereof, or a cell.

Suitable growth factors and cytokines that may be incorporated into thehydrogels of the invention include, but are not limited, to stem cellfactor (SCF), granulocyte-colony stimulating factor (G-CSF),granulocyte-macrophage stimulating factor (GM-CSF), stromal cell-derivedfactor-1, steel factor, VEGF, TGFβ, platelet derived growth factor(PDGF), angiopoeitins (Ang), epidermal growth factor (EGF), bFGF, HNF,NGF, bone morphogenic protein (BMP), fibroblast growth factor (FGF),hepatocye growth factor, insulin-like growth factor (IGF-1), interleukin(IL)-3, IL-1α, IL-1β, IL-6, IL-7, IL-8, IL-11, and IL-13,colony-stimulating factors, thrombopoietin, erythropoietin, fit3-ligand,and tumor necrosis factor α (TNFα). Other examples are described inDijke et al., “Growth Factors for Wound Healing”, Bio/Technology,7:793-798 (1989); Mulder G D, Haberer P A, Jeter K F, eds. Clinicians'Pocket Guide to Chronic Wound Repair. 4th ed. Springhouse, Pa.:Springhouse Corporation; 1998:85; Ziegler T. R., Pierce, G. F., andHerndon, D. N., 1997, International Symposium on Growth Factors andWound Healing: Basic Science & Potential Clinical Applications (Boston,1995, Serono Symposia USA), Publisher: Springer Verlag.

Examples of therapeutic or diagnostic agents which can be incorporatedin the hydrogels of the invention, or incorporated into the lipid basednanoparticles, e.g., a liposomes or a virosome, encapsulated by thehydrogels of the invention, include, but are not limited to, narcoticanalgesic drugs; salts of gold; corticosteroids; hormones; antimalarialdrugs; indole derivatives; pharmaceuticals for arthritis treatment;antibiotics, including Tetracyclines, Penicillin, Streptomycin andAureomycin; antihelmintic and canine distemper drugs, applied todomestic animals and large cattle, such, as, for example, phenothiazine;drugs based on sulfur, such, as sulfioxazole; antitumor drugs;pharmaceuticals supervising addictions, such as agents controllingalcohol addiction and agents controlling tobacco addiction; antagonistsof drug addiction, such, as methadone; weight controlling drugs; thyroidgland controlling drugs; analgesics; drugs controlling fertilization orcontraception hormones; amphetamines; antihypertensive drugs;antiinflammatories agents; antitussives; sedatives; neuromuscularrelaxants; antiepileptic drugs; antidepressants; antidisrhythmic drugs;vasodilating drugs; antihypertensive diuretics; antidiabetic agents;anticoagulants; antituberculous agents; antipsychotic agents; hormonesand peptides. It is understood that above list is not full and simplyrepresents the wide diversification of therapeutic agents that may beincluded in the compositions. In some embodiments, therapeutic agent isMitoxantrone, peptide, polyclonal antibody, monoclonal antibody, antigenbinding fragment of an antibody, protein (e.g. VEGF) or plasmid DNA.

Those of ordinary skill in the art will recognize numerous othertherapeutic or diagnostic agents that may be incorporated into thehydrogels of the invention, or into lipid based nanoparticles, e.g.,liposomes or virosomes, encapsulated by the hydrogels of the invention.Examples include a radiosensitizer, a steroid, a xanthine, abeta-2-agonist bronchodilator, an anti-inflammatory agent, an analgesicagent, a calcium antagonist, an angiotensin-converting enzymeinhibitors, a beta-blocker, a centrally active alpha-agonist, analpha-1-antagonist, an anticholinergic/antispasmodic agent, avasopressin analogue, an antiarrhythmic agent, an antiparkinsonianagent, an antiangina/antihypertensive agent, an anticoagulant agent, anantiplatelet agent, a sedative, an ansiolytic agent, a peptidic agent, abiopolymeric agent, an antineoplastic agent, a laxative, anantidiarrheal agent, an antimicrobial agent, an antifingal agent, avaccine, a protein, or a nucleic acid. In a further aspect, thepharmaceutically active agent can be coumarin, albumin, steroids such asbetamethasone, dexamethasone, methylprednisolone, prednisolone,prednisone, triamcinolone, budesonide, hydrocortisone, andpharmaceutically acceptable hydrocortisone derivatives; xanthines suchas theophylline and doxophylline; beta-2-agonist bronchodilators such assalbutamol, fenterol, clenbuterol, bambuterol, salmeterol, fenoterol;antiinflammatory agents, including antiasthmatic anti-inflammatoryagents, antiarthritis antiinflammatory agents, and non-steroidalantiinflammatory agents, examples of which include but are not limitedto sulfides, mesalamine, budesonide, salazopyrin, diclofenac,pharmaceutically acceptable diclofenac salts, nimesulide, naproxene,acetaminophen, ibuprofen, ketoprofen and piroxicam; analgesic agentssuch as salicylates; calcium channel blockers such as nifedipine,amlodipine, and nicardipine; angiotensin-converting enzyme inhibitorssuch as captopril, benazepril hydrochloride, fosinopril sodium,trandolapril, ramipril, lisinopril, enalapril, quinapril hydrochloride,and moexipril hydrochloride; beta-blockers (i.e., beta adrenergicblocking agents) such as sotalol hydrochloride, timolol maleate, esmololhydrochloride, carteolol, propanolol hydrochloride, betaxololhydrochloride, penbutolol sulfate, metoprolol tartrate, metoprololsuccinate, acebutolol hydrochloride, atenolol, pindolol, and bisoprololfumarate; centrally active alpha-2-agonists such as clonidine;alpha-1-antagonists such as doxazosin and prazosin;anticholinergic/antispasmodic agents such as dicyclomine hydrochloride,scopolamine hydrobromide, glycopyrrolate, clidinium bromide, flavoxate,and oxybutynin; vasopressin analogues such as vasopressin anddesmopressin; antiarrhythmic agents such as quinidine, lidocaine,tocainide hydrochloride, mexiletine hydrochloride, digoxin, verapamilhydrochloride, propafenone hydrochloride, flecainide acetate,procainamide hydrochloride, moricizine hydrochloride, and disopyramidephosphate; antiparkinsonian agents, such as dopamine, L-Dopa/Carbidopa,selegiline, dihydroergocryptine, pergolide, lisuride, apomorphine, andbromocryptine; antiangina agents and antihypertensive agents such asisosorbide mononitrate, isosorbide dinitrate, propranolol, atenolol andverapamil; anticoagulant and antiplatelet agents such as Coumadin,warfarin, acetylsalicylic acid, and ticlopidine; sedatives such asbenzodiazapines and barbiturates; ansiolytic agents such as lorazepam,bromazepam, and diazepam; peptidic and biopolymeric agents such ascalcitonin, leuprolide and other LHRH agonists, hirudin, cyclosporin,insulin, somatostatin, protirelin, interferon, desmopres sin,somatotropin, thymopentin, pidotimod, erythropoietin, interleukins,melatonin, granulocyte/macrophage-CSF, and heparin; antineoplasticagents such as etoposide, etoposide phosphate, cyclophosphamide,methotrexate, 5-fluorouracil, vincristine, doxorubicin, cisplatin,hydroxyurea, leucovorin calcium, tamoxifen, flutamide, asparaginase,altretamine, mitotane, and procarbazine hydrochloride; laxatives such assenna concentrate, casanthranol, bisacodyl, and sodium picosulphate;antidiarrheal agents such as difenoxine hydrochloride, loperamidehydrochloride, furazolidone, diphenoxylate hdyrochloride, andmicroorganisms; vaccines such as bacterial and viral vaccines;antimicrobial agents such as penicillins, cephalosporins, andmacrolides, antifungal agents such as imidazolic and triazolicderivatives; and nucleic acids such as DNA sequences encoding forbiological proteins, and antisense oligonucleotides.

Anti-cancer agents include alkylating agents, platinum agents,antimetabolites, topoisomerase inhibitors, antitumor antibiotics,antimitotic agents, aromatase inhibitors, thymidylate synthaseinhibitors, DNA antagonists, farnesyltransferase inhibitors, pumpinhibitors, histone acetyltransferase inhibitors, metalloproteinaseinhibitors, ribonucleoside reductase inhibitors, TNF alphaagonists/antagonists, endothelinA receptor antagonists, retinoic acidreceptor agonists, immuno-modulators, hormonal and antihormonal agents,photodynamic agents, and tyrosine kinase inhibitors.

Antibiotics include aminoglycosides (e.g., gentamicin, tobramycin,netilmicin, streptomycin, amikacin, neomycin), bacitracin, corbapenems(e.g., imipenem/cislastatin), cephalosporins, colistin, methenamine,monobactams (e.g., aztreonam), penicillins (e.g., penicillin G,penicillin V, methicillin, natcillin, oxacillin, cloxacillin,dicloxacillin, ampicillin, amoxicillin, carbenicillin, ticarcillin,piperacillin, mezlocillin, azlocillin), polymyxin B, quinolones, andvancomycin; and bacteriostatic agents such as chloramphenicol,clindanyan, macrolides (e.g., erythromycin, azithromycin,clarithromycin), lincomyan, nitrofurantoin, sulfonamides, tetracyclines(e.g., tetracycline, doxycycline, minocycline, demeclocyline), andtrimethoprim. Also included are metronidazole, fluoroquinolones, andritampin.

Enzyme inhibitors are substances which inhibit an enzymatic reaction.Examples of enzyme inhibitors include edrophonium chloride,N-methylphysostigmine, neostigmine bromide, physostigmine sulfate,tacrine, tacrine, 1-hydroxy maleate, iodotubercidin,p-bromotetramiisole, 10-(alpha-diethylaminopropionyl)-phenothiazinehydrochloride, calmidazolium chloride,hemicholinium-3,3,5-dinitrocatechol, diacylglycerol kinase inhibitor I,diacylglycerol kinase inhibitor II, 3-phenylpropargylamine,N^(o)-monomethyl-Larginine acetate, carbidopa, 3-hydroxybenzylhydrazine,hydralazine, clorgyline, deprenyl, hydroxylamine, iproniazid phosphate,6-MeO-tetrahydro-9H-pyrido-indole, nialamide, pargyline, quinacrine,semicarbazide, tranylcypromine,N,N-diethylaminoethyl-2,2-diphenylvalerate hydrochloride,3-isobutyl-1-methylxanthne, papaverine, indomethacind,2-cyclooctyl-2-hydroxyethylamine hydrochloride,2,3-dichloro-a-methylbenzylamine (DCMB), 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine hydrochloride, p-amino glutethimide,p-aminoglutethimide tartrate, 3-iodotyrosine, alpha-methyltyrosine,acetazolamide, dichlorphenamide, 6-hydroxy-2-benzothiazolesulfonamide,and allopurinol.

Antihistamines include pyrilamine, chlorpheniramine, andtetrahydrazoline, among others.

Anti-inflammatory agents include corticosteroids, nonsteroidalanti-inflammatory drugs (e.g., aspirin, phenylbutazone, indomethacin,sulindac, tolmetin, ibuprofen, piroxicam, and fenamates), acetaminophen,phenacetin, gold salts, chloroquine, D-Penicillamine, methotrexatecolchicine, allopurinol, probenecid, and sulfinpyrazone.

Muscle relaxants include mephenesin, methocarbomal, cyclobenzaprinehydrochloride, trihexylphenidyl hydrochloride, levodopa/carbidopa, andbiperiden.

Anti-spasmodics include atropine, scopolamine, oxyphenonium, andpapaverine.

Analgesics include aspirin, phenybutazone, idomethacin, sulindac,tolmetic, ibuprofen, piroxicam, fenamates, acetaminophen, phenacetin,morphine sulfate, codeine sulfate, meperidine, nalorphine, opioids(e.g., codeine sulfate, fentanyl citrate, hydrocodone bitartrate,loperamide, morphine sulfate, noscapine, norcodeine, normorphine,thebaine, nor-binaltorphimine, buprenorphine, chlomaltrexamine,funaltrexamione, nalbuphine, nalorphine, naloxone, naloxonazine,naltrexone, and naltrindole), procaine, lidocain, tetracaine anddibucaine.

Ophthalmic agents include sodium fluorescein, rose bengal, methacholine,adrenaline, cocaine, atropine, alpha-chymotrypsin, hyaluronidase,betaxalol, pilocarpine, timolol, timolol salts, and combinationsthereof.

Prostaglandins are art recognized and are a class of naturally occurringchemically related, long-chain hydroxy fatty acids that have a varietyof biological effects.

Anti-depressants are substances capable of preventing or relievingdepression. Examples of anti-depressants include imipramine,amitriptyline, nortriptyline, protriptyline, desipramine, amoxapine,doxepin, maprotiline, tranylcypromine, phenelzine, and isocarboxazide.

Trophic factors are factors whose continued presence improves theviability or longevity of a cell. Trophic factors include, withoutlimitation, platelet-derived growth factor (PDGP), neutrophil-activatingprotein, monocyte chemoattractant protein, macrophage-inflammatoryprotein, platelet factor, platelet basic protein, and melanoma growthstimulating activity; epidermal growth factor, transforming growthfactor (alpha), fibroblast growth factor, platelet-derived endothelialcell growth factor, insulin-like growth factor, glial derived growthneurotrophic factor, ciliary neurotrophic factor, nerve growth factor,bone growth/cartilage-inducing factor (alpha and beta), bonemorphogenetic proteins, interleukins (e.g., interleukin inhibitors orinterleukin receptors, including interleukin 1 through interleukin 10),interferons (e.g., interferon alpha, beta and gamma), hematopoieticfactors, including erythropoietin, granulocyte colony stimulatingfactor, macrophage colony stimulating factor and granulocyte-macrophagecolony stimulating factor; tumor necrosis factors, and transforminggrowth factors (beta), including beta-1, beta-2, beta-3, inhibin, andactivin.

Hormones include estrogens (e.g., estradiol, estrone, estriol,diethylstibestrol, quinestrol, chlorotrianisene, ethinyl estradiol,mestranol), anti-estrogens (e.g., clomiphene, tamoxifen), progestins(e.g., medroxyprogesterone, norethindrone, hydroxyprogesterone,norgestrel), antiprogestin (mifepristone), androgens (e.g., testosteronecypionate, fluoxymesterone, danazol, testolactone), anti-androgens(e.g., cyproterone acetate, flutamide), thyroid hormones (e.g.,triiodothyronne, thyroxine, propylthiouracil, methimazole, andiodixode), and pituitary hormones (e.g., corticotropin, sumutotropin,oxytocin, and vasopressin). Hormones are commonly employed in hormonereplacement therapy and/or for purposes of birth control. Steroidhormones, such as prednisone, are also used as immunosuppressants andanti-inflammatories.

The therapeutic or diagnostic agent can be an osteogenic protein.Accordingly, in some embodiments, the therapeutic or diagnostic agent isselected from the family of proteins known as the transforming growthfactors beta (TGF-β) superfamily of proteins, which includes theactivins, inhibins and bone morphogenetic proteins (BMPs). Mostpreferably, the active agent includes at least one protein selected fromthe subclass of proteins known generally as BMPs, which have beendisclosed to have osteogenic activity, and other growth anddifferentiation type activities. These BMPs include BMP proteins BMP-2,BMP-3, BMP-4, BMPS, BMP-6 and BMP-7, disclosed for instance in U.S. Pat.Nos. 5,108,922; 5,013,649; 5,116,738; 5,106,748; 5,187,076; and5,141,905; BMP-8, disclosed in PCT publication WO91/18098; and BMP-9,disclosed in PCT publication WO93/00432, BMP-10, disclosed in PCTapplication WO94/26893; BMP-11, disclosed in PCT application WO94/26892,or BMP-12 or BMP-13, disclosed in PCT application WO 95/16035; BMP-14;BMP-15, disclosed in U.S. Pat. No. 5,635,372; or BMP-16, disclosed inU.S. Pat. No. 5,965,403. Other TGF-β proteins, which can be used includeVgr-2, Jones et al., Mol. Endocrinol. 611961 (1992), and any of thegrowth and differentiation factors (GDFs), including those described inPCT applications WO94/15965; WO94/15949; WO95/01801; WO95/01802;WO94/21681; WO94/15966; WO95/10539; WO96/01845; WO96/02559 and others.Also useful in the invention can be BIP, disclosed in WO94/01557;HP00269, disclosed in JP Publication number: 7-250688; and BMP-14 (alsoknown as MP52, CDMP1, and GDF5), disclosed in PCT applicationWO93/16099. The disclosures of all of the above applications areincorporated herein by reference. Subsets of BMPs which can be usedinclude BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9,BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, andBMP18. Other osteogenic agents known in the art can also be used, suchas teriparatide (FORTEO™), CHRYSALIN®, prostaglandin E2, or LIM protein,among others.

The therapeutic or diagnostic agent, e.g., a protein or a peptide, canbe recombinantly produced, or purified from a protein composition. Theactive agent, if a TGF-β such as a BMP, or other dimeric protein, can behomodimeric, or can be heterodimeric with other BMPs (e.g., aheterodimer composed of one monomer each of BMP-2 and BMP-6) or withother members of the TGF-β superfamily, such as activins, inhibins andTGF-β1 (e.g., a heterodimer composed of one monomer each of a BMP and arelated member of the TGF-β superfamily). Examples of such heterodimericproteins are described for example in Published PCT Patent ApplicationWO 93/09229, the content of which is incorporated herein by reference.

The therapeutic or diagnostic agent can further refer to additionalagents such as the Hedgehog, Frazzled, Chordin, Noggin, Cerberus andFollistatin proteins. These families of proteins are generally describedin Sasai et al., (1994) Cell 791779-790 (Chordin); PCT PatentPublication WO94/05800 (Noggin); and Fukui et al., Devel. Biol. 159:1-31 (1993) (Follistatin). Hedgehog proteins are described inWO96/16668; WO96/17924; and WO95/18856. The Frazzled family of proteinsis a recently discovered family of proteins with high homology to theextracellular binding domain of the receptor protein family known asFrizzled. The Frizzled family of genes and proteins is described in Wanget al., Biol. Chem. 271:44684476 (1996). The active agent can alsoinclude other soluble receptors, such as the truncated soluble receptorsdisclosed in PCT patent publication WO95/07982. From the teaching ofWO95/07982, one skilled in the art will recognize that truncated solublereceptors can be prepared for numerous other receptor proteins. Theabove publications are hereby incorporated by reference herein.

The hydrogels of the invention may comprise cells. The cells amenable tobe encapsulated by the hydrogels of the invention include, but are notlimited to, stem cells (embryonic stem cells, mesenchymal stem cells,bone-marrow derived stem cells and hematopoietic stem cells),chrondrocytes progenitor cells, pancreatic progenitor cells, myoblasts,fibroblasts, keratinocytes, neuronal cells, glial cells, astrocytes,pre-adipocytes, adipocytes, vascular endothelial cells, hair follicularstem cells, endothelial progenitor cells, mesenchymal cells, neural stemcells and smooth muscle progenitor cells.

In some embodiments, the cell is a genetically modified cell. A cell canbe genetically modified to express and secrete a desired compound, e.g.,a bioactive agent, a growth factor, differentiation factor, cytokines,and the like. Methods of genetically modifying cells for expressing andsecreting compounds of interest are known in the art and easilyadaptable by one of skill in the art.

Differentiated cells that have been reprogrammed into stem cells canalso be used. For example, human skin cells reprogrammed into embryonicstem cells by the transduction of Oct3/4, Sox2, c-Myc and Klf4 (JunyingYu, et. al., Science, 2007, 318:1917-1920 and Takahashi K. et. al.,Cell, 2007, 131:1-12).

Cells useful for incorporation into the composition can come from anysource, e.g., a mammal. For example, the cell can be from a human, a rator a mouse. Human cells include, but are not limited to, human cardiacmyocytes-adult (HCMa), human dermal fibroblasts-fetal (HDF-f), humanepidermal keratinocytes (HEK), human mesenchymal stem cells-bone marrow,human umbilical mesenchymal stem cells, human hair follicular inner rootsheath cells, human umbilical vein endothelial cells (HUVEC), and humanumbilical vein smooth muscle cells (HUVSMC), human endothelialprogenitor cells, human myoblasts, human capillary endothelial cells,and human neural stem cells.

Exemplary rat and mouse cells include, but not limited to, RN-h (ratneurons-hippocampal), RN-c (rat neurons-cortical), RA (rat astrocytes),rat dorsal root ganglion cells, rat neuroprogenitor cells, mouseembryonic stem cells (mESC) mouse neural precursor cells, mousepancreatic progenitor cells, mouse mesenchymal cells and mouseendodermal cells.

In some embodiments, tissue culture cell lines can be used in thehydrogels described herein. Examples of cell lines include, but are notlimited to, C166 cells (embryonic day 12 mouse yolk), C6 glioma Cellline, HL1 (cardiac muscle cell line), AML12 (nontransforminghepatocytes), HeLa cells (cervical cancer cell line) and Chinese HamsterOvary cells (CHO cells).

An ordinary skill artisan in the art can locate, isolate and expand suchcells. In addition, the basic principles of cell culture and methods oflocating, isolation and expansion and preparing cells for tissueengineering are described in “Culture of Cells for Tissue Engineering”Editor(s): Gordana Vunjak-Novakovic, R. Ian Freshney, 2006 John Wiley &Sons, Inc., and Heath C. A., Trends in Biotechnology, 2000, 18:17-19,content of both of which is herein incorporated by reference in itsentirety.

In one embodiment, the biologic may be a peptide, e.g., a peptide havinga molecular weight of 250 kDa or less. In a further embodiment, thepeptide is an angiogenesis factor, e.g., FGF, VEGF, VEGFR, IGF, NRP-1,Ang1, Ang2, PDGF, PDGFR, TGF-β, endoglin, a TGF-β receptor, MCP-1,integrin, an integrin ligand (e.g., an RGD peptide), VE-cadherin, CD31,ephrin, plasminogen activator, plasminogen activator inhibitor-1, eNOS,COX-2, AC133, ID1 or ID3. In a specific embodiment, the peptideencapsulated by the hydrogels of the present invention is VEGF.

Biologics, such as polynucleotides, polypeptides, or other agents (e.g.,antigens) are purified and/or isolated. Specifically, as used herein, an“isolated” or “purified” nucleic acid molecule, polynucleotide,polypeptide, or protein, is substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.Purified compounds are at least 60% by weight (dry weight) the compoundof interest. The preparations herein can also be at least 75%, morepreferably at least about 90%, and most preferably at least about 99%,by weight the compound of interest. For example, a purified compound isone that is at least about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 98%, about 99%, or about 100% (w/w) of the desiredcompound by weight. Purity is measured by any appropriate standardmethod, for example, by column chromatography, thin layerchromatography, or high-performance liquid chromatography (HPLC)analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA)or deoxyribonucleic acid (DNA)) is free of the genes or sequences thatflank it in its naturally-occurring state. Purified also defines adegree of sterility that is safe for administration to a human subject,e.g., lacking infectious or toxic agents.

Similarly, by “substantially pure” is meant that a nucleotide,polypeptide, or other compound has been separated from the componentsthat naturally accompany it. Typically, the nucleotides and polypeptidesare substantially pure when they are at least about 60%, about 70%,about 80%, about 90%, about 95%, about 99%, or even 100%, by weight,free from the proteins and naturally-occurring organic molecules withthey are naturally associated. Examples include synthesized compounds,recombinant compounds (e.g., peptides, proteins, nucleic acids) orpurified compounds, such as purified by standard procedures includingchromatographic methods.

An “isolated nucleic acid” is a nucleic acid, the structure of which isnot identical to that of any naturally occurring nucleic acid, or tothat of any fragment of a naturally occurring genomic nucleic acidspanning more than three separate genes. The term covers, for example:(a) a DNA which is part of a naturally occurring genomic DNA molecule,but is not flanked by both of the nucleic acid sequences that flank thatpart of the molecule in the genome of the organism in which it naturallyoccurs; (b) a nucleic acid incorporated into a vector or into thegenomic DNA of a prokaryote or eukaryote in a manner, such that theresulting molecule is not identical to any naturally occurring vector orgenomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment,a fragment produced by polymerase chain reaction (PCR), or a restrictionfragment; and (d) a recombinant nucleotide sequence that is part of ahybridgene, i.e., a gene encoding a fusion protein. Isolated nucleicacid molecules according to the present disclosure further includemolecules produced synthetically, as well as any nucleic acids that havebeen altered chemically and/or that have modified backbones.

The therapeutic or diagnostic agent which may be encapsulated in thehydrogel of the invention or in a liposome encapsulated in the hydrogelof the invention, may be a STING adjuvant, a CRISPR-Cas 9 reagent and anadjuvant-loaded subcellular vesicle derived from disrupted cancer cells.

In one embodiment, the therapeutic or diagnostic agent may also be avaccine.

The polymers (e.g., alginate polymers) of the hydrogel may be about1-90% crosslinked, e.g., at least about 1%, about 10%, about 20%, about30%, about 40%, about 50%, about 60%, or about 70% crosslinked. Rangesintermediate to the recited values are also intended to be part of thisinvention. For example, the polymers of the hydrogel may be about 1% toabout 10%, about 7% to about 15%, about 12% to about 20%, about 15% toabout 30%, about 20% to about 40%, about 30% to about 50%, about 45% toabout 65% or about 50% to about 90% crosslinked.

The term “% crosslinked”, used interchangeably with the term“crosslinking density”, refers to the number of moles of click moietiesconjugated per moles of alginate monomers that have reacted with eachother.

The polymer (e.g., alginate) can be oxidized (e.g., highly oxidized),reduced, or be a mixture thereof. For example, a hydrogel of theinvention may comprise a mixture of polysaccharide polymers, e.g.,alginate polymers, that comprise algoxinol and algoxalate. In somecases, oxidized polymers or partially oxidized polymers arebiodegradable. For example, hydrogels comprising oxidized or partiallyoxidized alginate are biodegradable.

Therapeutic or diagnostic agent or a lipid based nanoparticle, e.g., aliposome or a virosome, encapsulating a therapeutic or diagnostic agentis released from the hydrogel of the invention in a sustained releasemanner, e.g., at a constant rate during a given number of hours, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23 or 24 hours; or days, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 days;or weeks, e.g., 1, 2, 3, 4, 5, 6, 7 or 8 weeks; or months, e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. When a therapeutic or adiagnostic agent or a lipid based nanoparticle, e.g., a liposome or avirosome, encapsulating a therapeutic or diagnostic agent is releasedfrom the hydrogel of the invention at a constant rate, the amount of thetherapeutic or diagnostic agent or the amount of lipid basednanoparticle released from the hydrogel in a given time period is aboutthe same, e.g., within about 0% to about 20%, of the amount of thetherapeutic or diagnostic agent or the lipid based nanoparticle releasedfrom the hydrogel during the time period immediately prior orimmediately after the given time period.

The rate of release of the therapeutic or diagnostic agent or of lipidbased nanoparticle encapsulating the therapeutic or diagnostic agent maybe modulated by changing one or more parameters during the preparationof the hydrogel. Such parameters include, but are not limited to, thedegree of click substitution; % polysaccharide oxidation; concentrationof the click conjugated polysaccharide during the cross-linking reaction(gelation); or the pH of the surrounding environment.

The degree of click substitution may be modulated by varying the numberof molar equivalents of the click reagent in the click conjugationreaction with the oxidized and reduced or a highly oxidizedpolysaccharide, e.g., algoxinol or algoxalate. For example, one mayincrease the number of click molecules conjugated to a polysaccharide ofthe invention by increasing the number of molar equivalents of the clickreagent in the click conjugation reaction. In another example, one maydecrease the number of click molecules conjugated to a polysaccharide ofthe invention by decreasing the number of molar equivalents of the clickreagent in the click conjugation reaction. In one embodiment, the clickconjugation reaction may comprise about 1 to about 2000 molarequivalents of a click reagent, e.g., about 1, about 5, about 10, about20, about 50, about 100, about 150, about 200, about 250, about 300,about 350, about 400, about 450, about 500, about 550, about 600, about650, about 700, about 750, about 800, about 850, about 900, about 950 orabout 1000 molar equivalents of the click reagent. Ranges intermediateto the recited values are also intended to be part of this invention.For example, the click conjugation reaction may comprise about 1 toabout 5, about 2 to about 10, about 5 to about 10, about 10 to about 50,about 40 to about 150, about 100 to about 400, about 300 to about 500,about 400 to about 1000, about 500 to about 1500 or about 1500 to about2000 molar equivalents of the click reagent.

In one embodiment, the polysaccharide, e.g., the oxidized and reduced ora highly oxidized polysaccharide, e.g., comprising algoxinol oralgoxalate, may comprise a degree of click substitution that is about0.01% to about 90%, e.g., about 0.01%, about 0.05%, about 0.1%, about0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45% or about 90%. Rangesintermediate to the recited values are also intended to be part of thisinvention. For example, the polysaccharide may be about 0.01% to about0.05%, about 0.01% to about 1%, about 0.1% to about 5%, about 5% toabout 15%, about 10% to about 20%, about 15% to about 25%, about 20% toabout 35%, about 30% to about 40% or about 35% to about 50% clicksubstituted.

One may also modulate the number of click molecules conjugated to apolysaccharide of the invention by modulating % oxidation of thepolysaccharide. For example, higher % oxidation of the polysaccharideprior to its reduction, e.g., with ammonia borane, or, prior to itsfurther oxidation, e.g., with sodium chlorite, may create additionalaldehyde moieties, which, upon their conversion to alcohol moieties uponreduction, or to carboxylic acid moieties upon further oxidation, mayserve as sites for click reagent conjugation.

Modulating the degree of click substitution of the polysaccharide of theinvention may allow modulating the time to gelation, or the time ittakes the click conjugated polysaccharide to cross-link and to produce ahydrogel. For example, a higher degree of click substitution may lead toshorter gelation times.

Modulating the degree of click substitution also allows modulating thecross-linking density and the average diameter of pores in the resultinghydrogel, i.e., mesh size of the resulting hydrogel. For example, ahigher degree of click substitution may lead to smaller average porediameter, or mesh size of the hydrogel, which may lead to a decreasedrate of release of the therapeutic or diagnostic agent or a lipid basednanoparticle, e.g., a liposome or a virosome, comprising the therapeuticor diagnostic agent. A higher degree of click substitution may also leadto longer retention of the lipid based nanoparticles comprising thetherapeutic or diagnostic agent in the hydrogel.

In yet another example, the concentration of the polysaccharide of theinvention during the cross-linking reaction may be varied. For example,a higher concentration of a polysaccharide, e.g., a click conjugatedpolysaccharide, in the click conjugation reaction, may lead to smallermesh size of the resulting hydrogel, which may, in turn, cause adecreased rate of release of the therapeutic or diagnostic agent or alipid based nanoparticle, e.g., a liposome or a virosome, comprising thetherapeutic or diagnostic agent from the hydrogel of the invention. Ahigher degree of click substitution may also lead to longer retention oflipid based nanoparticles comprising the therapeutic or diagnostic agentin the hydrogel. In some embodiments, the concentration of clickconjugated polysaccharide during hydrogel formation, e.g., concentrationof the alginate material present in the solution immediately prior toformation of the hydrogel, may be from about 0.01% to about 50% w/v,e.g., about 0.01% to about 10% w/v, about 0.1% to about 5% w/v, about 1%to about 15% w/v, about 10% to about 30% w/v, about 12% to about 35%w/v, about 15% to about 25% w/v, about 20% to about 45% w/v or about 35%to about 50% w/v.

Hydrogels of the present invention, e.g., hydrogels comprising alginate,such as hydrogels comprising algoxinol and/or algoxalate, are notdegraded by endogenous enzymes present in a host or a subject, e.g., ahuman. The hydrogels of the invention may be chemically degraded, e.g.,hydrolyzed when exposed to acidic or alkaline condition. Acidicconditions comprise a pH 6.5 or lower, while alkaline conditionscomprise a pH 8 or higher. Without wishing to be bound by a theory, itis believed that the rate of chemical degradation of the hydrogels ofthe invention is pH dependent. For example, the rate of chemicaldegradation of the hydrogels of the invention at pH 2 is higher than atpH 6, and the rate of chemical degradation of the hydrogels of theinvention at pH 8 is higher than at pH 12.

When the hydrogel of the invention is exposed to acidic or alkalineconditions, the cargo comprised in the hydrogel, e.g., a therapeutic ordiagnostic agent, or a lipid based nanoparticle, e.g., a liposome or avirosome, encapsulating a therapeutic or diagnostic agent, is releasedfrom the hydrogel at an increased rate as compared to its rate ofrelease at neutral pH, e.g., pH of 7.4. For example, because theintratumoral environment may be characterized by an acidic pH of 6.5 orlower, the rate of cargo release from the hydrogels of the invention maybe increased in the intratumoral environment, thereby allowing sustainedand targeted delivery of anti-cancer agents.

V. Lipid Based Nanoparticles Encapsulated in the Hydrogels of theInvention

The present invention also provides a drug delivery compositioncomprising a lipid based nanoparticle encapsulating a therapeutic ordiagnostic agent and a hydrogel of the invention encapsulating theliposome. The term “lipid based nanoparticle”, as used herein, refers toany nanoparticle that comprises lipids and that may be used for deliveryfor a therapeutic or diagnostic agent to a subject. In one embodiment,the lipid based nanoparticle is a liposome. In another embodiment, thelipid based nanoparticle is a virosome.

Delivering a therapeutic or a diagnostic agent in a liposome may bedesirable because liposomes may provide protection of such agentsagainst systemic enzymatic degradation, or may be used to mimic antigenpresenting cells (APCs). However, there are challenges associated withliposomal drug delivery. For example, the composition of a liposome mayhave a big effect on the loading efficiency, efficacy of the therapeuticor diagnostic agent encapsulated by the liposome and the systemictoxicity of the therapeutic or diagnostic agent encapsulated by theliposome. Accordingly, there is a need for sustained, localizedliposomal drug delivery that may reduce systemic toxicity and providelocalization of drug cargos to target tissues. However, currentlyavailable liposome delivery compositions cannot retain liposomal cargos,with release of the liposomal cargos occurring within hours or days. Thecompositions currently available in the art are also unable to deliverintact liposomes to target locations, e.g., in cases intact liposomesare required for cargos to cross the cell membrane for cytosolicdelivery.

It has been surprisingly discovered that the reduced and/or highlyoxidized polysaccharides of the invention, e.g., polysaccharidescomprising algoxinol and/or algoxalate, e.g., made using a methoddescribed herein, are particularly useful for preparing hydrogels thatmay be used to encapsulate liposomes. Specifically, it has beensurprisingly discovered that the hydrogels of the invention, e.g.,hydrogels prepared from alginate comprising algoxinol and/or algoxalateconjugated to click reagents, can retain intact liposomes over a longperiod of time and can deliver intact liposomes to a desired location ina subject, e.g., to a cytosol of a cell.

As used herein, the term “intact liposome” includes a liposome thatretains its size, e.g., average diameter, while it is encapsulated by ahydrogel, or while it is in the process of being delivered to a desiredlocation within a host or a subject, e.g., a human. An intact liposomeis one that resembles the therapeutic input and does not coalesce withother liposomes to generate polydisperse liposomes of a larger size,e.g., larger average diameter, nor fragment into smaller liposomes ormicelles.

The liposome may be delivered to a desired location, e.g., to a cytosolof a cell, in an intact form after the hydrogel encapsulating theliposome is degraded inside a host, e.g., a subject, such as a human. Inone example, the hydrogel comprises alginate, e.g., algoxinol oralgoxalate, which is not susceptible to degradation by a host endogenousenzyme. Because alginate, e.g., algoxinol or algoxalate, is notsusceptible to degradation by a host endogenous enzyme, a hydrogelcomprising alginate is capable of retaining a liposome for a prolongedperiod of time. For example, a liposome of the invention may remainencapsulated in the hydrogel for at least 5 days, at least 10 days, atleast 15 days, at least 20 days, at least 25 days, at least 30 days, atleast 35 days, at least 40 days, at least 45 days, at least 50 days, atleast 55 days, at least 60 days, at least 65 days, at least 70 days, atleast 75 days or at least 80 days.

A liposome that remains intact during delivery to a desired locationwithin a host or a subject is a liposome that retains it average size,e.g., an average diameter, during the delivery. For example, the averagediameter of an intact liposome that has been encapsulated by a hydrogelof the invention is within about 50%, e.g., about 49%, about 48%, about47%, about 46%, about 45%, about 44%, about 43%, about 42%, about 41%,about 40%, about 39%, about 38%, about 37%, about 36%, about 35%, about34%, about 33%, about 32%, about 31%, about 30%, about 29%, about 28%,about 27%, about 26%, about 25%, about 24%, about 23%, about 22%, about21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%,about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2% or about1%, of the average diameter of the same liposome prior to encapsulation,or within about 25%, e.g., about 24%, about 23%, about 22%, about 21%,about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%,about 7%, about 6%, about 5%, about 4%, about 3%, about 2% or about 1%,of the average diameter of a standard. The standard may be a liposomeprepared in the same manner as the liposome used for encapsulation witha hydrogel. Size of a liposome may be measured by any method known toone of skill in the art, for example, by dynamic light scattering (DLS).

A liposome useful in the context of the present invention may be anyliposome known in the art that may be used to deliver a therapeutic ordiagnostic agent. For example, the liposome may be a neutral liposome,e.g., a liposome comprising uncharged lipids (Niosome). Examples of suchlipids include, but are not limited to,1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol (CHOL).The liposome may also be a charged liposome, e.g., a cationic or ananionic liposome comprising, e.g., a positively charged or a negativelycharged lipids. Non-limiting examples of positively charged lipidsinclude N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammoniummethyl-sulfate (DOTAP) and hydrogenated soy phosphatidilcholine (HydroSoy PC). Non-limiting examples of negatively charged lipids include1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG). In someembodiments, the liposome useful in the context of the present inventionis a therapeutic liposome or a diagnostic liposome, e.g., a liposomecomprising a therapeutic or diagnostic agent as described above.

A virosome is a drug or vaccine delivery mechanism consisting ofunilamellar phospholipid membrane (either a mono- or bi-layer) vesicleincorporating virus derived proteins to allow the virosomes to fuse withtarget cells. Virosomes are not able to replicate but are purefusion-active vesicles. A virosome useful in the context of the presentinvention may be any virosome known in the art that may be used todeliver any virus derived proteins. In some embodiments, the virosomemay comprise a protein derived from an influenza virus, e.g.,hemagglutinin or neuraminidase. In some embodiments, a virosome is atherapeutic or a diagnostic virosome, e.g., a virosome comprising atherapeutic or diagnostic agent as described above. In some embodiments,a virosome comprises a vaccine.

VI. Pharmaceutical Compositions of the Invention

For administration to a subject, the hydrogels of the invention can beprovided in pharmaceutically acceptable (e.g., sterile) compositions.Accordingly, another aspect described herein is a pharmaceuticalcomposition comprising a hydrogel and a pharmaceutically acceptablecarrier. These pharmaceutically acceptable compositions comprise ahydrogel formulated together with one or more pharmaceuticallyacceptable carriers (additives) and/or diluents. As described in detailbelow, the pharmaceutical compositions of the present disclosure can bespecifically formulated for administration in solid or liquid form,including those adapted for the following: (1) oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),lozenges, dragees, capsules, pills, tablets (e.g., those targeted forbuccal, sublingual, and/or systemic absorption), boluses, powders,granules, pastes for application to the tongue; (2) parenteraladministration, for example, by subcutaneous, intramuscular, intravenous(e.g., bolus or infusion) or epidural injection as, for example, asterile solution or suspension, or sustained-release formulation; (3)topical application, for example, as a cream, ointment, or acontrolled-release patch or spray applied to the skin; (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam; (5) sublingually; (6) ocularly; (7) transdermally; (8)transmucosally; or (9) nasally. Additionally, compounds can be implantedinto a patient or injected using a drug delivery system. See, forexample, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236(1984); Lewis, ed. “Controlled Release of Pesticides andPharmaceuticals” (Plenum Press, New York, 1981); U.S. Pat. No.3,773,919; and U.S. Pat. No. 35 3,270,960, content of all of which isherein incorporated by reference.

As used herein, the term “pharmaceutically acceptable” or“pharmacologically acceptable” refers to those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio. Moreover, for animal (e.g., human)administration, it will be understood that compositions should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biological Standards.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, disintegrating agents, binders, sweetening agents, flavoringagents, perfuming agents, protease inhibitors, plasticizers,emulsifiers, stabilizing agents, viscosity increasing agents, filmforming agents, solubilizing agents, surfactants, preservative andantioxidants can also be present in the formulation. The terms such as“excipient”, “carrier”, “pharmaceutically acceptable carrier” or thelike are used interchangeably herein.

The hydrogel of the invention comprising the therapeutic agent can bedelivered to an in vivo locus in a subject. Exemplary in vivo lociinclude, but are not limited to site of a wound, trauma or disease. Thehydrogel can be delivered to the in vivo locus by, for example,implanting the compositions into a subject. Hydrogels that are to beimplanted, i.e., implantable devices, can additionally include one ormore additives. Additives can be resolving (biodegradable) polymers,mannitol, starch sugar, inosite, sorbitol, glucose, lactose, saccharose,sodium chloride, calcium chloride, amino acids, magnesium chloride,citric acid, acetic acid, hydroxyl-butanedioic acid, phosphoric acid,glucuronic acid, gluconic acid, poly-sorbitol, sodium acetate, sodiumcitrate, sodium phosphate, zinc stearate, aluminium stearate, magnesiumstearate, sodium carbonate, sodium bicarbonate, sodium hydroxide,polyvinylpyrolidones, polyethylene glycols, carboxymethyl celluloses,methyl celluloses, starch or their mixtures.

The implantable device can have virtually any regular or irregular shapeincluding, but not limited to, spheroid, cubic, polyhedron, prism,cylinder, rod, disc, or other geometric shape. Accordingly, in someembodiments, the implant is of cylindrical form from about 0.5 to about10 mm in diameter and from about 0.5 to about 10 cm in length.Preferably, its diameter is from about 1 to about 5 mm and length fromabout 1 to about 5 cm.

In some embodiments, the implantable device is of spherical form. Whenthe implantable device is in a spherical form, its diameter can rangefrom about 0.5 to about 50 mm in diameter. In some embodiments, aspherical implant's diameter is from about 5 to about 30 mm. Preferablythe diameter is from about 10 to about 25 mm.

In some embodiments, the hydrogel of the present invention may be usedto prepare a drug delivery device or a drug depot. The drug deliverydevice may also be biodegradable and refillable. Refillablebiodegradable drug delivery devices are described, e.g., inPCT/US2015/024540 and U.S. application Ser. No. 14/878,578, the entirecontents of each of which are hereby incorporated herein by reference.The refillable biodegradable drug delivery devices comprise a targetrecognition moiety capable of interacting with a target conjugated to adrug refill.

In some embodiments, the hydrogel of the invention used to prepare arefillable biodegradable drug delivery device may comprise apolysaccharide conjugated to at least two click reagents belonging totwo different click pairs as described above. For example, thepolysaccharide may comprise a first click reagent that functions as atarget recognition moiety for the drug delivery device by reacting withits click pair conjugated to the drug refill. The polysaccharide mayalso comprise a second click reagent that functions as a cross-linkingagent as described above, or that functions to conjugate a moiety, e.g.,a therapeutic agent, to the polysaccharide. In one embodiment, the firstclick reagent and the second click reagent do not cross-react, i.e., donot react with each other, but only react with their click pairs.

In other embodiments, the hydrogel of the invention may be used toprepare a drug refill for the refillable biodegradable drug deliverydevice as described above. The hydrogel used to prepare the drug refillmay comprise a polysaccharide conjugated to at least two click reagentsas described above, where at least one click reagent may function as atarget by reacting with its click pair conjugated to the drug deliverydevice.

VII. Methods of Treatment Using Hydrogels of the Invention

The present invention also relates to methods of treating a subject inneed thereof. The methods comprise administering to the subject aneffective amount of a hydrogel, a dosage form, a pharmaceuticalcomposition or an implantable drug delivery device of the invention.

As used herein, the term “administer” refers to the placement of acomposition into a subject by a method or route which results in atleast partial localization of the composition at a desired site suchthat desired effect is produced. A compound or composition describedherein can be administered by any appropriate route known in the artincluding, but not limited to, oral or parenteral routes, includingintravenous, intramuscular, subcutaneous, transdermal, airway (aerosol),pulmonary, nasal, rectal, and topical (including buccal and sublingual)administration.

Exemplary modes of administration include, but are not limited to,injection, infusion, instillation, inhalation, or ingestion. “Injection”includes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and intrasternal injection and infusion. Inpreferred embodiments, the compositions are administered by intravenousinfusion or injection.

Administration can also be by transmucosal or transdermal means. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the compounds are formulated into ointments, salves, gels, or creams asgenerally known in the art.

In some embodiments, administration includes implanting a composition,e.g., a hydrogel, described herein in a subject.

The term “therapeutically effective amount”, as used herein, means thatamount of a compound, material, or composition comprising a compounddescribed herein which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells in a subject ata reasonable benefit/risk ratio applicable to any medical treatment.Thus, “therapeutically effective amount” means that amount which, whenadministered to a subject for treating a disease, is sufficient toeffect such treatment for the disease.

Determination of an effective amount is well within the capability ofthose skilled in the art. Generally, the actual effective amount canvary with the specific compound, the use or application technique, thedesired effect, the duration of the effect and side effects, thesubject's history, age, condition, sex, as well as the severity and typeof the medical condition in the subject, and administration of otherpharmaceutically active agents. Accordingly, an effective dose ofcompound described herein is an amount sufficient to produce at leastsome desired therapeutic effect in a subject.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of use or administration utilized.

The effective dose can be estimated initially from cell culture assays.A dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC₅₀ (i.e., theconcentration of the therapeutic which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Levels in plasmacan be measured, for example, by high performance liquid chromatography.The effects of any particular dosage can be monitored by a suitablebioassay.

Generally, the term “treatment” or “treating” is defined as theapplication or administration of a therapeutic agent to a patient, orapplication or administration of a therapeutic agent to an isolatedtissue or cell line from a patient, said patient having a disease, asymptom of disease or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease, the symptoms of disease or thepredisposition toward disease. Thus, treating can include suppressing,inhibiting, preventing, treating, or a combination thereof. Treatingrefers, inter alia, to increasing time to sustained progression,expediting remission, inducing remission, augmenting remission, speedingrecovery, increasing efficacy of or decreasing resistance to alternativetherapeutics, or a combination thereof. “Suppressing” or “inhibiting”,refers, inter alia, to delaying the onset of symptoms, preventingrelapse to a disease, decreasing the number or frequency of relapseepisodes, increasing latency between symptomatic episodes, reducing theseverity of symptoms, reducing the severity of an acute episode,reducing the number of symptoms, reducing the incidence ofdisease-related symptoms, reducing the latency of symptoms, amelioratingsymptoms, reducing secondary symptoms, reducing secondary infections,prolonging patient survival, or a combination thereof. In one embodimentthe symptoms are primary, while in another embodiment, symptoms aresecondary. “Primary” refers to a symptom that is a direct result of adisorder, e.g., diabetes, while, secondary refers to a symptom that isderived from or consequent to a primary cause. Symptoms may be anymanifestation of a disease or pathological condition.

Accordingly, as used herein, the term “treatment” or “treating” includesany administration of a compound described herein and includes: (i)preventing the disease from occurring in a subject which may bepredisposed to the disease but does not yet experience or display thepathology or symptomatology of the disease; (ii) inhibiting the diseasein an subject that is experiencing or displaying the pathology orsymptomatology of the diseased (i.e., arresting further development ofthe pathology and/or symptomatology); or (iii) ameliorating the diseasein a subject that is experiencing or displaying the pathology orsymptomatology of the diseased (i.e., reversing the pathology and/orsymptomatology).

By “treatment”, “prevention” or “amelioration” of a disease or disorderis meant delaying or preventing the onset of such a disease or disorder,reversing, alleviating, ameliorating, inhibiting, slowing down orstopping the progression, aggravation or deterioration the progressionor severity of a condition associated with such a disease or disorder.In one embodiment, the symptoms of a disease or disorder are alleviatedby at least 5%, at least 10%, at least 20%, at least 30%, at least 40%,or at least 50%.

Efficacy of treatment is determined in association with any known methodfor diagnosing the disorder. Alleviation of one or more symptoms of thedisorder indicates that the compound confers a clinical benefit. Any ofthe therapeutic methods described to above can be applied to anysuitable subject including, for example, mammals such as dogs, cats,cows, horses, rabbits, monkeys, and most preferably, humans.

As used herein, the term “subject” includes any subject who may benefitfrom being administered a hydrogel or an implantable drug deliverydevice of the invention. The term “subject” includes animals, e.g.,vertebrates, amphibians, fish, mammals, non-human animals, includinghumans and primates, such as chimpanzees, monkeys and the like. In oneembodiment of the invention, the subject is a human.

The term “subject” also includes agriculturally productive livestock,for example, cattle, sheep, goats, horses, pigs, donkeys, camels,buffalo, rabbits, chickens, turkeys, ducks, geese and bees; and domesticpets, for example, dogs, cats, caged birds and aquarium fish, and alsoso-called test animals, for example, hamsters, guinea pigs, rats andmice.

In one embodiment, the present invention provides a method for treatingear related disorders. The method comprises administering to a subjectin need thereof a hydrogel, or a dosage form for treating ear relateddisorders, such as otitis. Such dosage forms are administered into anexternal auditory canal of a subject in need thereof. The dosage formsmay comprise a variety of therapeutic agents, e.g., at least oneantibiotic. The drug delivery devices for delivering therapeutic agentsinto an ear are described, e.g., in US 2014/0107423, the entire contentsof which are hereby incorporated herein by reference.

In another embodiment, the present invention also provides a hydrogel,or a dosage form for treating eye related disorders. The methodcomprises administering into an eye of a subject in need thereof ahydrogel, or a dosage form of the invention. The hydrogel, or a dosageform of the invention may comprise at least one therapeutic agent.Non-limiting examples of the therapeutic agents that may be used in anophthalmic dosage form include, e.g., antibiotics, corticoids, localanaesthetics, decongestants, non-steroidal antiphlogistics, virustatics,antiseptics, cortisone, anti-allergic active substances, prostaglandinanalogues, active substances from the active substance class ofantihistamines and corticosteroids, anti-allergic active substances,pantothenic acid derivatives, non-steroidal anti-inflammatory drugs,vascoconstrictors and/or anti-glaucoma active substances in apharmaceutically effective concentration. Suitable dosage forms foradministration into an eye are described, e.g., in US 20150139973, theentire contents of which are hereby incorporated herein by reference.

The present invention also provides methods for treating chronicischemia in a subject in need thereof. The method comprisesadministering to the subject an effective amount of a hydrogel, a dosageform, or an implantable device, of the invention. In an embodiment, thehydrogel, the dosage form, or an implantable device of the inventioncomprises VEGF.

It has been previously found that delivering VEGF locally to the site ofischemia may increase the half-life of VEGF in a subject's body andreduce side treatment related side effects. Accordingly, in oneembodiment, the hydrogel, the dosage form or the implantable device isadministered locally to the site of ischemia. In some embodiments, thehydrogel, the dosage form or the implantable device is administered tothe tissue to be engrafted before and/or after transplantation.

The present invention also provides methods for regenerating a tissue ina subject in need thereof. The method comprises administering to thesubject an effective amount of a hydrogel, a dosage form, or animplantable device, of the invention that further comprises a cell. Forexample, the cell may be a mammalian cell, and the tissue may be amammalian tissue. In some embodiments, the mammalian cell is of the sametype as the tissue to be regenerated. In other embodiments, themammalian cell is a stem cell. Embodiments of the methods providedherein include contacting a mammalian tissue with a hydrogel, a dosageform, or an implantable device of the invention that further comprises acell.

In another example, a method for regenerating a tissue in a subjectcomprises providing a hydrogel, a dosage form, or an implantable devicedescribed herein, wherein the hydrogel comprises a cell immobilizedwithin the hydrogel (i.e., the cell remains within the hydrogel for anextended period of time without exiting the hydrogel). The methodincludes contacting a tissue with the hydrogel, wherein the cell isimmobilized within the hydrogel. In some embodiments, the cell is aprogeny cell. In some embodiments, the hydrogel remains stable and doesnot allow for host cell infiltration.

In one embodiment, the hydrogels described herein are useful as animmunoprotective barrier, e.g., for pancreatic islet transplantation. Insome cases, pancreatic islet transplantation is a treatment fordiabetes, e.g., Type I diabetes. Transplanted cells, such as islets, canbe destroyed by immune reactions, and the hydrogels of the invention arecapable of encapsulating cells, such as islet cells, prior toimplantation/injection of the hydrogel. This way, the hydrogels serve asan immunoprotective barrier in a subject and minimize immune rejectionof transplanted cells and tissues.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting. The entire contents of all ofthe references cited throughout this application are hereby expresslyincorporated herein by reference.

EXAMPLES Example 1: Aldehydes Present in Alginates can React with CargoProteins and Click Reagents

Alginates of high and low molecular weight were oxidized by reactionwith sodium periodate. To this end, alginate with MW of ˜265 kDa (MVG orProtanol LF 20/40, FMC Technologies; I-1G or 1-8, Kimica Corp.) and MWof ˜32 kDa (VLVG, FMC Technologies) was dissolved in ddH₂O at theconcentration of 1% w/v. Sodium periodate (Sigma-Aldrich) was added tothe solution in an amount that was based on the percent molar ratio tomoles of alginate monomer. The solution was then allowed to reactovernight in the dark at room temperature. Sodium chloride was added tothe solution to the concentration of 0.3 M, and the oxidized product waspurified by Tangential Flow Filtration (TFF) or dialyzed using 12-14 kDaMWCO dialysis tubing (Spectrum Labs) overnight with extensive waterchanges against a decreasing salt gradient from 150 mM to 0 mM NaCl indiH₂O.

Oxidized alginates containing 0%-50% aldehydes were incubated with VEGF.Percent change in soluble, un-denatured, VEGF (2% w/v alginate, 4 mcg/mLVEGF) was measured 5 days after incubation by ELISA (R&D Systems Cat#DVE00). As is shown in FIG. 2a , an increase in the % of aldehydes isdirectly correlated with % change in VEGF, with alginates containing 50%aldehydes resulting in 50% change in VEGF. The data demonstrates thataldehydes incubated with oxidized alginates can react with VEGF.

Oxidized alginates containing 20%-50% aldehydes were also conjugatedwith click reagents tetrazine (Tz) and norbornene (Nb). The oxidizedalginates showed significant color changes after resuspension at roomtemperature, indicating an aldehyde concentration dependence ondegradation. Shown in FIG. 2b is a picture of vials containing theoxidized alginates conjugated to Tz and Nb after resuspension. Shownfrom left to right are:

-   -   alginate with 20% aldehydes conjugated to Tz;    -   alginate with 20% aldehydes conjugated to Nb;    -   alginate with 30% aldehydes conjugated to Tz;    -   alginate with 30% aldehydes conjugated to Nb;    -   alginate with 40% aldehydes conjugated to Tz;    -   alginate with 40% aldehydes conjugated to Nb;    -   alginate with 50% aldehydes conjugated to Tz;    -   alginate with 50% aldehydes conjugated to Nb.

The data demonstrate that aldehydes can also react with click reagentsto form colored products and imines in a manner dependent on aldehydeconcentration. These color shifts represent undesirable changes to theclick moieties. Imines can be hydrolyzed back to aldehydes in acidicconditions.

Example 2. Reduction of Alginate Aldehydes

The purpose of this experiment was to determine the content of residualaldehydes in oxidized alginates that have been reacted with sodiumborohydride (NaBH₄), ammonia borane (BH₃NH₃) and sodium chlorite(NaClO₂). As is illustrated in FIG. 3a , both sodium borohydride andammonia borane react with aldehydes present in alginates to formalcohols, while sodium chlorite reacts with aldehydes to form carboxylicacids.

Alginate was oxidized with sodium periodate, such that it contained5-50% residual aldehydes. The oxidized alginate was then reacted withsodium borohydride, ammonia borane and sodium chlorite, and % ofresidual aldehydes were quantified using qNMR after each reaction.

Sodium periodate oxidation of alginate was carried out using theprocedure as described in Example 1.

To reduce oxidized alginate with sodium borohydride, the alginatesolution was treated with sodium borohydride (SB, Sigma-Aldrich) at themolar ratio of SB to alginate oxidized monomers of >1.5:1. The reactionwas allowed to proceed overnight at room temperature. Sodium chloridewas added to the solution to reach the concentration of 0.3 M, and theSB treated product was purified by Tangential Flow Filtration (TFF) ordialyzed using 12-14 kDa MWCO dialysis tubing (Spectrum Labs) overnightwith extensive water changes against a decreasing salt gradient from 150mM to 0 mM NaCl in diH₂O. The solution containing reduced alginate wasthen frozen and lyophilized to dryness.

To reduce oxidized alginate with ammonia borane, the alginate solutionwas treated with ammonia borane (AB) complex (Sigma-Aldrich) at themolar ratio of AB to alginate aldehydes of >1.5:1. The reaction wasallowed to proceed overnight at room temperature. Sodium chloride wasadded to the solution to reach the concentration of 0.3M, and the ABtreated product was purified by Tangential Flow Filtration (TFF) ordialyzed using 12-14 kDa MWCO dialysis tubing (Spectrum Labs) overnightwith extensive water changes against a decreasing salt gradient from 150mM to 0 mM NaCl in diH₂O. The solution containing reduced alginate wasthen frozen and lyophilized to dryness.

To further oxidize oxidized alginate, the alginate solution was treatedwith sodium chlorite (SC) at the molar ratio of SC to alginate aldehydesof >1.5:1. Prior to addition of sodium chlorite, dimethylsufoxide (DMSO;Sigma-Aldrich) was added to the solution at the molar ratio of DMSO tosodium chlorite of 5:1 and mixed until the solution became homogenous.The reaction was allowed to proceed overnight at room temperature.Sodium chloride was then added to the solution to reach theconcentration of 0.3M, and the SC treated product was purified byTangential Flow Filtration (TFF) or dialyzed using 12-14 kDa MWCOdialysis tubing (Spectrum Labs) overnight with extensive water changesagainst a decreasing salt gradient from 150 mM to 0 mM NaCl in diH₂O.The solution containing the highly oxidized alginate was then frozen andlyophilized to dryness.

FIG. 3b is a bar graph showing % residual oxidation in oxidizedalginates that have been further reduced with ammonia borane or sodiumborohydride, or that have been further oxidized with sodium chlorite.The data indicate that ammonia borane is superior to sodium borohydridein its ability to reduce aldehydes and produces alginate containing thelowest level of residual aldehydes. This effect is particularly evidentfor alginates with higher levels of oxidation. The data also indicatethat sodium chlorite oxidation of aldehydes does not reach completion atthe “initial DMSO reaction conditions” (1:1 water:DMSO). The residualaldehydes may be eliminated by changing the reaction conditions to 33:1water:DMSO, as shown in FIG. 6 k.

Example 3. Reductive Processing of Oxidized Alginates Reduces the Effectof Aldehydes on Cargo Proteins

The goal of this experiment was to evaluate the effect of aldehydespresent in oxidized alginate on the biological activity of VEGF. To thisend, alginates were oxidized by reaction with sodium periodate toproduce oxidized alginates containing 0%-50% aldehydes. The oxidizedalginates were then reacted with sodium borohydride (NaBH₄), ammoniaborane (BH₃NH₃) and sodium chlorite (NaClO₂). The different alginateswere incubated with VEGF at 37° C. in PBS containing 1% BSA at alginateconcentration of 20 mg/mL and VEGF concentration of 4 μg/mL. Percentchange in soluble, un-denatured, VEGF (2% w/v alginate, 4 mcg/mL VEGF)was measured 5 days after incubation in EBM cell culture media or in asolution containing 1% BSA by ELISA (R&D Systems Cat #DVE00).

The data is shown in FIGS. 4a and 4b . FIG. 4a shows % change in VEGFmeasured after 5 days of incubation in EBM. The data indicates that foroxidized alginates that have not been further processed, the % change inVEGF is directly correlated with the percent of aldehydes contained inalginate (see first 7 bars from the left, labeled as “0”, “1”, “5”,“10”, “15”, “25” and “50”). The % change in VEGF reaches 100% foralginates containing 25% and 50% of aldehydes. The data also indicatesthat % change in VEGF is significantly reduced for oxidized alginatesthat have been reacted with ammonia borane (see bars labeled as “AB5”,“AB10”, “AB15”, “AB25” and “AB50”), with the highest % change of about60% in VEGF seen for the alginate containing 15% of aldehydes. Reductionwith sodium borohydride is not as effective at protecting VEGF, withhigher % change in VEGF seen for each studied alginate material (seebars labeled as “SB5”, “SB10”, “SB15”, “SB25” and “SB50”). Oxidationwith sodium chlorite resulted in significant % change measured for allstudied samples (see bars labeled as “SC5”, “SC10”, “SC15”, “SC25” and“SC50”), due to the residual aldehydes maintained by reactioninefficiency at the time (initial DMSO reaction conditions).

FIG. 4b shows % change in VEGF measured after 5 days of incubation in 1%BSA solution. The data indicates that % change in VEGF can reach morethan 30% for oxidized alginate materials that have not been furtherprocessed (see first 7 bars from the left, labeled as “0% Ox”, “1% Ox”,“5% Ox”, “10% Ox”, “15% Ox”, “25% Ox” and “50% Ox”). Reductiveprocessing with ammonia borane (see bars labeled as “AB5”, “AB10”,“AB15”, “AB25” and “AB50”) or sodium borohydride (see bars labeled as“SC5”, “SC10”, “SC15”, “SC25” and “SC50”) reduced % change in VEGF, withsodium borohydride being more effective than ammonia borane. Oxidativeprocessing with sodium chlorite (initial DMSO reaction conditions)resulted in an increase in % change in VEGF (see bars labeled as “SC5”,“SC10”, “SC15”, “SC25” and “SC50”), as compared to controls.

The data shown in FIGS. 4a and 4b indicate that there are differences inthe ability of variously processed alginates to protect protein cargo.

Example 4. The Degree of Click Reagent Substitution is Increased forOxidized Alginates after Reaction with Sodium Chlorite

The purpose of this experiment was to determine if the degree of clickreagent conjugation with a click reagent may be increased followingreaction of oxidized alginates with sodium chlorite. As shown in FIG. 1a, click reagents may be typically conjugated to alginates via thecarboxylate moiety present in the glucuronic acid in an alginate,resulting in one click molecule per alginate monomer. Following alginateoxidation with sodium periodate, two aldehyde reagents are generated peralginate monomer, which may also be conjugated to a click reagent via animine bond (FIG. 1b ). This conjugation of click reagents to oxidizedalginates is sub-optimal because of residual aldehydes that remain inthe alginate following conjugation that may result in toxicity anddamage to cargo, as well as degradation of the click reagents (seeexamples 1 and 3). Further, the imine bonds between the aldehydes andthe click reagents are easily hydrolyzable, resulting in the loss ofclick reagents from the alginate. However, as shown in FIG. 1c , furtheroxidation of aldehydes present in oxidized alginates converts thesealdehydes to carboxylic acids provides two additional sites for clickconjugation.

In order to determine if further oxidation of oxidized alginates canincrease click conjugation, alginate reacted with sodium periodate only,or alginate oxidized with sodium periodate (HighOx alginate) and furtheroxidized with sodium chlorite (alginate containing algoxalate) wereconjugated with click reagents tetrazine (Tz) and norbornene (Nb), andthe relative amounts of conjugated tetrazine and norbornene werecompared. To this end, alginate was oxidized using sodium periodate andSC using the procedure described in Examples 1 and 2. These productswere then modified with either 1-bicyclo[2.2.1]hept-5-en-2-ylmethanamine(Norbornene Methanamine; TCI) or 3-(p-benzylamino)-1,2,4,5-tetrazine.First, the SC treated alginate was dissolved in stirred buffercontaining 0.1 M MES, 0.3 M NaCl, pH 6.5 at the concentration of 0.5%w/v. Next, N-hydroxysuccinimide (NHS; Sigma-Aldrich) and1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC;Sigma-Aldrich) were added in 5× molar excess of the carboxylic acidgroups present on the alginate. Either norbornene (Nb) or tetrazine (Tz)was then added at defined molar ratios of Nb or Tz to alginate monomerto yield Alg-N or Alg-T, respectively. The coupling reaction was stirredat room temperature overnight, and the product is purified by TangentialFlow Filtration (TFF) or dialyzed using 12-14 kDa MWCO dialysis tubing(Spectrum Labs) overnight with extensive water changes against adecreasing salt gradient from 150 mM to 0 mM NaCl in diH₂O. The purifiedAlg-N and Alg-T polymers were then treated with activated charcoal,sterile filtered (0.22 mm), and freeze-dried. This resulted in purifiedAlg-N or Alg-T polymers with various degrees of substitution of theavailable carboxylic acid groups of alginate.

FIG. 5a shows pictures of vials containing HighOx alginates conjugatedto Tz and Nb at day 0 (top panel) and HighOx Sc alginates conjugated toTz and Nb at day 0 (bottom panel). Tz- and Nz-conjugated material isnominally pink/red and slightly off clear, respectively. FIG. 5b showsrelative amounts of Tz present in HighOx and HighOx SC alginates,measured by NMR. The data indicate that there is a 1.4 to 2.3 times moreTz molecules conjugated to HighOx SC alginate, as compared to the HighOxalginate, for alginates containing 20-50% oxidation, indicating that thealginate containing algoxalate has an extended conjugation potential.

Example 5. Characterization of Click Conjugated Oxidized Alginates

The purpose of this experiment was to characterize the properties ofclick conjugated HighOx SC alginate material and compare it with theproperties of click conjugated HighOx alginate material. The stabilityof both alginate materials was investigated by UV-Vis spectral analysis.The data for Tz-conjugated materials is shown in FIGS. 6a and 6b . FIG.6a shows a UV-Vis spectrum of Tz-conjugated HighOx alginate materialwith 20-50% oxidation at day 28 (top panel) and a UV-Vis spectrum ofTz-conjugated HighOx SC material with 20% and 30% conjugation at day 14.FIG. 6b also shows pictures of vials containing these Tz-conjugatedHighOx and the HighOx SC alginate materials. The data indicates thattetrazine conjugated HighOx alginate material loses the characteristicTz peak at 515 nm at 28 days, while this peak is still present at day 14in HighOx SC alginate material containing 20% and 30% oxidation. Thesedata suggest that the sodium chlorite processed tetrazine conjugatedmaterial exhibits greater stability in solution.

The data for Nb-conjugated materials is shown in FIGS. 6c and 6d . FIG.6c shows a UV-Vis spectrum of Nb-conjugated HighOx alginate materialwith 20-50% oxidation at day 28 (top panel) and a UV-Vis spectrum ofNb-conjugated HighOx SC material with 20%-50% conjugation at day 14.FIG. 6d also shows pictures of vials containing these Nb-conjugatedHighOx and the HighOx SC alginate materials. The data indicates that aplateau at 475 nm is produced in HighOx alginate material at day 28,while this peak is absent in the HighOx SC alginate material at day 14.The HighOx SC alginate material is also characterized by a reducedabsorption at 300 nm. These data suggest that the Sodium Chloriteprocessed norbornene conjugated material exhibits greater stability insolution.

The UV-Vis spectra for Tz-conjugated and Nb-conjugated HighOx (20% and50% aldehyde) ammonia borane (AB), sodium borohydride (SB), and sodiumchlorite (SC) alginate materials at day 15 (4% w/v solutions stored atroom temperature) are shown in FIG. 6j . The spectra indicate that after15 days the peak at 515 nm for Tz-conjugated HighOx material isretained, and that there is no absorption at 300 nm for theNb-conjugated HighOx material. This data indicates that reduction orfurther oxidation of the alginate aldehydes confers greater stability ofthe click conjugated moieties in solution.

The Tz-conjugated and Nb-conjugated HighOx and HighOx SC materials werealso investigated by quantitative NMR to determine their aldehydecontent. Quantification of the aldehyde content was performed on aVarian 400 MHz by comparing the proton peaks generated by sodiumperiodate at >˜5.1 ppm to an internal standard of Dimethylmalonic Acid(DMMA; 6H@ 1.3 pmm; Sigma-Aldrich). Samples were prepared at 15 mg/mLalginate and 2.5 mg/mL DMMA in deuterium oxide (D20; Sigma-Aldrich).

The results are shown in FIGS. 6e, 6f, 6g and 6h . Specifically, FIGS.6e and 6f show NMR spectra of HighOx alginate material with 50%oxidation conjugated to Nb and Tz, respectively. FIGS. 6g and 6h showNMR spectra of of HighOx SC alginate material with 50% aldehydeconjugated to Nb and Tz, respectively. The data indicate that there is asignificant peak broadening in HighOx aldehyde alginate material,indicating that this material is more dynamic. The NMR spectra forHighOx alginate material also contain peaks indicating presence ofresidual aldehydes. In contrast, the NMR spectra for HighOx SC alginatematerial show sharp peaks, indicating greater definition in the HighOxSC material, and a near absence of residual aldehydes. The absence ofresidual aldehydes in HighOx SC alginate material is also confirmed bythe FTIR analysis, with the FTIR spectrum shown in FIG. 6i . Overall,the data indicate that click-conjugated HighOx SC alginate material isless dynamic, more chemically stable and contains only minor amounts ofresidual aldehydes.

The reaction conditions of oxidized alginate with sodium chlorite wereevaluated. Initial reaction conditions used to generate the data shownin FIG. 3b used a mixture of 1:1 water:DMSO as a solvent, where DMSO wasused as a scavenger for the hypochlorous acid (HOC1), a by-product. Bychanging the DMSO content in the mixture to 33:1 water:DMSO, thereaction was driven further to completion. The NMR spectrum for theHighOx SC alginate material produced at optimal conditions is shown inFIG. 6k demonstrating that there are not any significant residualaldehydes in the material with 20% and 50% oxidation after furtherprocessing the aldehydes with sodium chlorite under these conditions.

Example 6. Gelation of Click Conjugated Oxidized Alginates

Click conjugated HighOx and HighOx SC material with 20-50% oxidation wasinvestigated for the ability to form a hydrogel. Preparations ofcomplimentary degrees of oxidation (e.g. 20% aldehyde-TZ and 20%aldehyde-Nb) were made at 10% (w/v), mixed and allowed to gel for 20minutes. FIGS. 8a and 8b demonstrate that periodate oxidized, reduced(AB, SB) and further oxidized (SC) materials degrade as a function oftime in solution (e.g. dialysis residence time), which limits hydrogelformation. Thus, in order to potentiate gelation, the process has beenmodified to replace dialysis with tangential flow filtration (TFF) inorder to minimize solution residence time.

FIG. 8c demonstrates that the solubility of the HighOx and HighOx SCmaterial increases as a function of % oxidation and is highest for theHighOx SC material.

Example 7. In Vitro Degradation and Solubility of Oxidized and ReducedAlginates

The purpose of this experiment was to investigate the degradation andsolubility of oxidized alginates with and without click conjugationafter further reductive processing (i.e., reaction with sodiumborohydride or ammonia borane) or oxidative processing (i.e., reductionwith sodium chlorite).

Click conjugated alginate material (MVG alginate, 280 kDa) was preparedby reacting alginate with sodium periodate, followed by precipitationwith tetrahydrofuran (THF). The precipitated material was then reducedby reacting it with either sodium borohydride or ammonia borane, orfurther oxidized by reacting it with sodium chlorite, and furtherdialyzed for 1 day. It was then conjugated with Tz or Nb, and dialyzedagain for 1 day.

Control alginate materials not conjugated with clicks (VLVG alginate, 30kDa) was prepared by reacting alginate with sodium periodate, followedby a 3-day dialysis. The dialyzed material was then reduced by reactingit with either sodium borohydride or ammonia borane, or further oxidizedby reacting it with sodium chlorite, and further dialyzed for 3 days.

The MVG and VLVG alginate materials were then incubated at 37° C. for upto 29 days, and were analyzed by gel permeation chromatography (GPC) todetermine the average molecular weight of the alginate material. To thisend, samples were prepared in PBS at the concentration of the alginatematerial of 2 mg/mL. The samples were then analyzed using Agilent 1260HPLC equipped with G4000PW×1 and G5000PW×1 columns in series. Analysiswas conducted using 100 μL injections, with 0.5 mL/min flow rate, 0.1 MSodium Nitrate (Sigma-Aldrich) mobile phase, with column and detectortemperatures at 35° C. Samples were compared to a calibrated PEOstandard (34 kDa; Agilent Technologies) using triple detection.

The results are shown in FIGS. 7a and 7b . Specifically, FIG. 7a showsdegradation of Tz-conjugated and Nb-conjugated MVG alginate with 20%oxidation (left panel) and 50% oxidation (right panel). The dataindicates that reductively processed MVG alginates (AB, SB) and furtheroxidized (SC) materials all exhibit degradability.

FIG. 7b shows degradation of VLVG alginate containing 0%-50% oxidationat 37° C. for non-processed alginate (upper left panel), alginatereductively processed with ammonia borane (upper right panel), alginatereductively processed with sodium borohydride (lower left panel) andalginate processed with sodium chlorite (lower right panel). The dataindicate that reduced and further oxidized materials exhibit degradationprofiles similar to the periodate oxidized material controls.

FIG. 7c is a picture of vials containing 50% w/v solutions of MVG (280kDa) materials with 20% oxidation processed with either ammonia boraneor sodium chlorite. Unoxidized VLVG (30 kDa) at 50% (w/v) is provided asa control. Unoxidized MVG is only soluble to ˜5% w/v, while unoxidizedVLVG is soluble to ˜10% w/v. Surprisingly, this photograph shows thatboth ammonia borane and sodium chlorite processed materials (having amolecular weight similar to VLVG, see FIG. 8a ) are readily soluble at50% w/v. The data indicate that Tz-conjugated and Nb-conjugated MVGmaterials that have been further oxidized with sodium chlorite are themost soluble.

Example 8. Upper Limit of Click Conjugation for Tetrazine and Norbornene

The purpose of this experiment was to determine the upper limit of clickconjugation for the VLVG material prepared as described in Example 7.The MVG material, also prepared as described in Example 7, uses 250molar equivalents of the click material in the conjugation reaction toobtain approximately 5% degree of substitution (DS), defined as thenumber of click moieties per monomer unit, with the upper limit for Tzbeing approximately 7.5%, and for Nb being 20%. In this experiment, theunoxidized VLVG material was reacted with different equivalents of Tz orNb, and the % DS was measured by qNMR. The data is displayed in thetable below. Norbornene NMR peaks broaden significantly at higher molarequivalence, thus values for norbornene are assumed to be over reported(OR).

Equivalents of Click Reagent % DS by qNMR 600 eq tetrazine 12.0 600 eqnorbornene 37.1 (OR) 900 eq tetrazine 12.9 900 eq norbornene 72.9 (OR)1200 eq tetrazine 21.8 1200 eq norbornene 97.0 (OR)

Tetrazine conjugation was also measured using UV-Vis spectrometry, asshown in FIG. 8. The data indicates that the degree of substitution fortetrazine is linear for the reaction with 600-1200 tetrazineequivalents. These data indicate that greater degrees of substitutioncan be obtained using lower molecular weight alginates and that theupper limit has not yet been reached. This experiment is extended to thereduced and further oxidized materials to determine the upper limit oftheir conjugation potential, and thus crosslinking density.

Example 9. Effect of Aldehydes Present in Oxidized Alginate on CellViability

The goal of this experiment was to determine the relative cell viabilityin the presence of aldehydes present in oxidized alginates. To this end,alginate was oxidized with sodium periodate to generate aldehydecontaining alginate materials with 0-50% oxidation. Subsequently, 10 mgof this material was directly incubated for 2 days with 5×10⁵ mouseleukemia cells (ATCC CCL-219, n=1) in DMEM with 10% horse serum. Theviable cell number was quantified using MUSE Cell Count and ViabilityAssay Kit. The data, normalized to 0% oxidation, is shown in the tablebelow and also graphically in FIG. 9.

Sample (% oxidation) 2-day viable cell count Relative cell viability  0%ox 7.91 × 10⁵ 1.00  5% ox 8.06 × 10⁵ 1.02 15% ox 8.16 × 10⁵ 1.03 25% ox6.78 × 10⁵ 0.86 50% ox 2.58 × 10⁵ 0.33The data indicates that cell viability is not affected by aldehydes upto 15% oxidation, and starts to decline rapidly at 25% and 50%oxidation.

Example 10. Solubility of Oxidized Alginates

The purpose of this experiment was to compare the solubility of oxidizedalginate with click conjugation to the solubility of non-oxidizedalginate. FIG. 10 is a bar graph showing solubility (% w/v) forunoxidized alginate with molecular weight of 250 kDa (MVG); unoxidizedalginate with molecular weight of 30 kDa (VLVG); and alginate (MVG) withmolecular weight of 30 kDa (VLVG) that was oxidized to 20%, and thenfurther oxidized by using sodium clorite and conjugated with clicks (Tzor Nb) to produce a final product with a molecular weight of about 30kDa. The VLVG material used in this experiment is shown in vial 5 ofFIG. 7c . The click-conjugated and sodium processed alginate materialused in this experiment is shown in vials 3 and 4 of FIG. 7 c.

The data presented in FIG. 10 indicate that unoxidized MVG is onlysoluble to ˜5% w/v, unoxidized VLVG is soluble to ˜10% w/v; andclick-conjugated sodium chlorite processed alginate is soluble to ˜50%.The data demonstrate that alginate containing algoxalate, and, to aslightly lesser extent, alginate containing algoxalol, is characterizedby a Significantly Increased Solubility as Compared to UnoxidizedAlginate.

Example 11. Influence of Solubility of Alginate on the CrosslinkingPotential

The purpose of this experiment was to determine the upper limit of Tzconjugation for unoxidized alginate with molecular weights of 250 kDa(MVG) and 30 kDa (VLVG). MVG material is more viscous and less solublethan the VLVG material due to its higher molecular weight. Thisexperiment was conducted using the experimental procedure as describedin Example 8.

FIG. 11a is a bar graph showing the upper limit of Tz conjugationachieved for MVG and VLVG materials. The data in FIG. 11a demonstratesthat less soluble MVG material may be conjugated to a maximum of ˜500molar equivalents of Tz, while the more soluble VLVG material may beconjugated to a maximum of ˜2500 molar equivalents of Tz. Accordingly,increased solubility (due to lower molecular weight) of the VLVGmaterial allows for greater degree of click substitution. Thisconjugation potential may be further extended through the use ofalginate containing algoxanol and/or algoxalate due to its even lowerviscosity.

FIG. 11b is a graph showing degree of substitution of the VLVG materialas a function of molar equivalents of Tz. The material studied was VLVGreacted with 500, 1000, 1500, 2000 and 2500 molar equivalents of Tz. Itdemonstrates that the upper limit of Tz conjugation for VLVG material isachieved at ˜2500 equivalents of Tz.

Example 12. Influence of Alginate Concentration and Degree of ClickSubstitution on the Kinetics of Gelation of Click-Conjugated AlginateMaterials

The purpose of this experiment was to study the kinetics of gelation ofclick conjugated alginate material as a function of alginateconcentration, degree of oxidation and degree of click substitutionusing rheology measurements. This experiment utilized non-oxidized VLVGmaterial conjugated with Nb and Tz using conjugation reactions thatcontained 500, 1500 and 2500 molar equivalents of Nb or Tz). Thisexperiment also utilized MVG material that was oxidized to 10% or 20%oxidation using sodium periodate, reductively processed with ammoniaborane and then conjugated with Nb and Tz using 250 molar equivalents ofNb and Tz in the conjugation reaction. Also used in this experiment wasLF 20/40 alginate material which was oxidized to 20% oxidation usingsodium periodate, reductively processed with ammonia borane and thenconjugated to Nb or Tz using 1000 molar equivalents of Nb and Tz in theconjugation reaction.

Gelation of the click conjugated alginate material was determined bymeasuring the value of the elastic modulus G′ using rheologymeasurements. To this end, Alg-N and Alg-T solutions at desired finalconcentrations (5%, 10%, 15% or 20% w/v in PBS) were mixed at a 1:1ratio and directly pipetted onto the bottom plate of a TA InstrumentsARG2 rheometer equipped with 20 mm flat upper plate geometry and a400-micron geometry gap. A Peltier cooler was used to control thetemperature for temperature dependent experiments, and a water reservoircover was placed over the gel to prevent the hydrogel from drying duringtesting. Hydrogel samples were subjected to 1% strain at 1 Hz, and thestorage and loss moduli (G′ and G″) were monitored for 1 hour.

FIG. 12a is a graph showing the increase in the elastic modulus G′versus time for different studied VLVG materials. This data effectivelydemonstrate how long it takes for different materials to gelate, with aplateau indicating that a gel-like state has been achieved. The dataindicates that increasing degree of click substitution leads to morerapid gelation.

FIG. 12b is a bar graph showing the value of the elastic modulus G′ fordifferent studied VLVG materials. The bar graph shown in black on theleft and labeled “2% 1:3 MVG:VLVG” corresponds to control material,which is calcium crosslinked 2% w/v solution of 1:3 mixture of MVG:VLVG.

FIG. 12c is a bar graph showing the value of mesh size for differentstudied VLVG materials. The bar graph shown in black on the left andlabeled “2% 1:3 MVG:VLVG” corresponds to control material, which is acalcium crosslinked 2% w/v solution of 1:3 mixture of MVG:VLVG.

The data shown in FIGS. 12b and 12c indicate that increasing the degreeof click substitution leads to different rheological properties of thematerial, as manifested in the increased elastic modulus G′ and to areduction in pore size.

FIG. 12d is a graph showing the increase in the elastic modulus G′versus time for MVG material that was oxidized to 10% oxidation, reducedwith ammonia borane and then conjugated with Nb or Tz using 250 molarequivalents of Nb and Tz. Hydrogels were produced at the concentrationof click conjugated reduced alginate of 5% w/v, 10% w/v or 15% w/v. FIG.12e is a graph showing the increase in the elastic modulus G′ versustime for MVG material that was oxidized to 20% oxidation, reduced withammonia borane and then conjugated with Nb or Tz using 250 molarequivalents of Nb and Tz. Hydrogels were produced at the concentrationof click conjugated reduced alginate of 5% w/v, 10% w/v, 15% w/v or 20%w/v. FIG. 12f is a graph showing the increase in the elastic modulus G′versus time for LF 20/40 material that was oxidized to 20% oxidation,reduced with ammonia borane and then conjugated with Nb or Tz using 1000molar equivalents of Nb and Tz. Hydrogels were produced at theconcentration of click conjugated reduced alginate of 5% w/v, 10% w/v,15% w/v or 20% w/v.

The data in FIG. 12d , FIG. 12e and FIG. 12f indicate that increasingthe concentration of the reduced alginate reduces the time of gelationof the click conjugated alginates. The results demonstrate that it ispossible to modulate the gelation process of click conjugated alginatesby varying the degree of alginate oxidation, the relative amount ofclick reagents and the concentration of alginate in the clickconjugation reaction.

Example 13. Influence of the Degree of Click Substitution on the ProteinRelease Rate

The purpose of this experiment was to investigate the influence of thedegree of click substitution on the release rates of various proteinsencapsulated in click alginate hydrogels. This experiment utilizednon-oxidized VLVG and MVG material conjugated with Nb or Tz produced asdescribed in Example 11. Specifically, non-oxidized VLVG material wasconjugated with Nb or Tz at 250, 500, 1000, 1500, 2000 and 2500 molarequivalents of Nb or Tz to produce Nb and Tz conjugated alginate (Alg-Nand Alg-T). Non-oxidized MVG material was conjugated with Nb or Tz at250 molar equivalents of Nb or Tz. This experiment also utilizedproteins with different molecular weights, such as insulin (MW of ˜3.5kDa), bovine serum albumin (BSA, MW ˜67 kDa) and IgG (MW ˜150 kDa).

Protein release curves were evaluated by encapsulating insulin labeledwith fluorescein (Sigma-Aldrich), bovine serum albumin (BSA;Sigma-Aldrich), and human Imuunoglobulin G (IgG; Sigma-Aldrich) in clickconjugated alginate. Samples were prepared by first separatelydissolving freeze-dried Alg-N and Alg-T polymers of various degrees ofsubstitution to final desired concentration (4% and 5% w/v) in PBS. Theprotein of interest was added and mixed with the Alg-N solution at theratio of protein:alginate of 1:10 (Alg-N+Alg-T, v/v). This solution wasthen thoroughly mixed with the Alg-T solution, and the mixture wasallowed to gel for at least 30 minutes. Samples were created by adding 1mL of PBS to each gel and incubating at 37° C. Samples were collected atvarious timepoints with replacement of the supernatant at eachtimepoint. Protein content in the supernatant was quantified against astandard curve using a plate reader with fluorescence excitation at 492nm and emission at 518 nm.

FIG. 13a is a graph showing cumulative release (in micrograms) offluorescein conjugated insulin from hydrogels produced usingclick-conjugated VLVG materials with different degrees of clickconjugation. FIG. 13b is a graph showing the non-plateau region of thecurve in FIG. 13a corresponding to 2500 molar equivalents of Nb or Tzand 5% concentration of the Alg-N and Alg-T during hydrogel formation(2500 eq 5% VLVG) and the linear fit. FIG. 13c is a graph showingcumulative release (in micrograms) of fluorescein conjugated BSA fromhydrogels produced using click-conjugated VLVG materials with differentdegrees of click conjugation. FIG. 13d is a graph showing thenon-plateau region of the curve in FIG. 13c corresponding to 2500 molarequivalents of Nb or Tz and 5% concentration of the Alg-N and Alg-Tduring hydrogel formation (2500 eq 5% VLVG) and the linear fit. FIG. 13eis a graph showing cumulative release (in micrograms) of fluoresceinconjugated IgG from hydrogels produced using click-conjugated VLVGmaterials with different degrees of click conjugation. FIG. 13f is agraph showing the non-plateau region of the curve in FIG. 13ecorresponding to 2500 molar equivalents of Nb or Tz and 5% concentrationof the Alg-N and Alg-T during hydrogel formation (2500 eq 5% VLVG) andthe linear fit. FIG. 13g is a graph showing cumulative release (inmicrograms) of fluorescein conjugated insulin, BSA and IgG fromhydrogels produced by Ca²⁺ mediated crosslinking.

The data shown in FIGS. 13a-13g demonstrate that protein release profilefrom alginate hydrogels may be influenced by the degree of clicksubstitution of the alginate. Specifically, the data shows that a higherdegree of click substitution reduces the “initial burst” of proteinsfrom the hydrogel. Using alginates with a higher degree of clicksubstitution allows for the production of hydrogels with smaller “meshsize”, which, in turn, allows achieving longer protein release times.For example, as evidenced by FIGS. 13b, 13d and 13f , alginates havingthe highest degree of click substitution (2500 equivalents), producehydrogels that afford linear protein release rates over several days. Incontrast, hydrogels produced from other materials afford non-linear,“burst” protein release over hours, not days. Examples of othermaterials are described, for example, in K S Anseth, et. al., Biomed.Mater. Res. A, 2009, 90: 720-729; PP Kundu, et. al., CarbohydratePolymers, 2014, 112: 627-637; T. Bal, et. al., J. Biomed. Mater. Res.Part A, 2014. 102A: 487-495; C. E. Schmidt, et. al. Biomaterials, 2005,26: 125-135; Y. M. Lee, et. al., Macromol. Research, 2006. 14: 87-93; C.P. Covas, et. al., Mat. Sci. App. 2011. 2: 509-520; W. F. Mieler, et.al., Trans. Am. Ophtalmol. Soc., 2008. 106: 206-214; W. M. Tian, et.al., Controlled Release, 2005. 102: 13-22.

Example 14. Influence of the Degree of Alginate Oxidation and AlginateConcentration on the Protein Release Rate

The purpose of this experiment was to investigate the influence of thedegree of alginate oxidation and alginate concentration during thegelation process on the release rates of various proteins encapsulatedin click alginate hydrogels. This experiment utilized LF 20/40 alginatematerial that was initially oxidized by sodium periodate to 5% or 10%oxidation as described in Example 1. Subsequently, the oxidized LF 20/40alginate was either reductively processed with ammonia borane (AB) orfurther oxidized with sodium chlorite (SC) using the procedure describedin Example 2. This material was then conjugated with Nb or Tz using 2000molar equivalents of Nb or Tz and the procedure described in Example 4.Subsequently, this material was used to produce hydrogels and toencapsulate insulin or IgG at 5% or 10% w/v alginate concentration.Protein release profiles were monitored as described in Example 13.

FIG. 14a is a graph showing cumulative release (in micrograms) offluorescein conjugated insulin from hydrogels produced usingclick-conjugated LF 20/40 alginate at the concentration of 5% w/v thatwas oxidized to 5% or 10% and then reductively processed with AB.

FIG. 14b is a graph showing cumulative release (in micrograms) offluorescein conjugated insulin from hydrogels produced usingclick-conjugated LF 20/40 alginate at the concentration of 5% w/v thatwas oxidized to 5% or 10% and then oxidatively processed with SC.

FIG. 14c is a graph showing cumulative release (in micrograms) offluorescein conjugated IgG from hydrogels produced usingclick-conjugated LF 20/40 alginate at the concentration of 5% w/v thatwas oxidized to 5% or 10% and then reductively processed with AB.

FIG. 14d is a graph showing cumulative release (in micrograms) offluorescein conjugated IgG from hydrogels produced usingclick-conjugated LF 20/40 alginate at the concentration of 5% w/v thatwas oxidized to 5% or 10% and then oxidatively processed with SC.

The data in FIGS. 14a-14d indicate that modulation of protein releaseprofiles may be achieved by varying the degree of alginate oxidation forAb and SC processed material and the concentration of alginate materialduring the gelation process.

Example 15. Effect of Aldehydes Present in Oxidized Alginate on ClickMoieties

The goal of this experiment was to determine the stability of clickmoieties conjugated to alginate in the presence of aldehydes. To thisend, non-oxidized MVG material was conjugated to Nb or Tz in aconjugation reaction that used 250 equivalents of the click material,and was subsequently exposed to 2.3% of gluteraldehyde. The vials wereobserved for up to 69 hours for color change. FIG. 15a is a series ofpictures of glass vials containing 4% MVG material conjugated to Nb(left vial) and Tz (right vial) in the presence of 2.3% gluteraldehyde,taken after 0 minutes, 40 minutes, 21.5 hours and 67.5 hours. FIG. 15bis a series of pictures of glass vials containing water as control (leftvial) or 2% MVG material conjugated to DBCO (middle vial) or azide(right vial), taken after 0 minutes, 40 minutes, 20 hours and 69 hours.The data in FIG. 15a indicate that degradation of Nb and Tz is observedafter 40 minutes of exposure to aldehydes. Degradation of DBCO and azidetakes longer, but may be observed after 20 hours, as evidenced by thedata in FIG. 15b . Because aldehydes are generated upon oxidation ofalginate, this experiment demonstrates that click moieties conjugated toalginate will be expected to degrade upon oxidation of alginate.Therefore, to maintain click moiety stability, the aldehydes must beeither reductively eliminated, e.g., using reduction with ammoniaborane, or oxidatively eliminated, e.g., using further oxidation withsodium chlorite.

Example 16. Encapsulation and Retention of Liposomes by AlginateHydrogels

The purpose of this experiment was to investigate the ability ofhydrogels produced from click conjugated alginates to encapsulateliposomes and to provide sustained and localized delivery of intactliposomes in vivo. This experiment utilized commercially availableliposomes prepared from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)and cholesterol (CHOL), at the ratio of DOPC/CHOL of 55:45 and totallipid concentration of 50 mM. The liposomes also contained thefluorescent dye 1,1′-Dioctadecyl-3,3,3′,3′-TetramethylindocarbocyaninePerchlorate (Dil) at the concentration of 0.5 mM (0.47 mg/mL) and had anaverage diameter of 133 nm. The liposomes were encapsulated in hydrogelsprepared from non-oxidized MVG material by crosslinking in the presenceof CaSO4 and liposomes at the alginate concentration of 2% or 5% w/v.The liposomes were also encapsulated in hydrogels prepared fromnon-oxidized MVG material that was conjugated to Nb or Tz using 250molar equivalents of Nb or Tz and allowed to gel in the presence ofliposomes at the alginate concentration of 5% w/v. Finally, theliposomes were also encapsulated in hydrogels prepared from clickconjugated gelatin prepared as described in, e.g., Koshy et al.,Advanced Healthcare Materials 2016, Vol. 5, Issue 5, pages 541-547.Liposome release from the hydrogels was monitored in vitro by measuringthe increase in Dil fluorescence in the supernatant. Supernatant proteincontent was quantified against a standard curve using a platereader withfluorescence excitation at 550 nm and emission at 580 nm.

FIG. 16a is a graph showing cumulative release (% load) of liposomesfrom Ca²⁺ cross-linked alginate and from non-oxidized click conjugatedalginate over the period of 50 days. FIG. 16b is a graph showingcumulative release (% load) of liposomes from Ca²⁺ cross-linked alginatehydrogel, non-oxidized click conjugated alginate hydrogel and clickgelatin hydrogel. These results indicate that quantitative release ofliposomes from click gelatin hydrogel is observed after 30 days. Incontrast, alginate hydrogels were more effective in retaining liposomes.Specifically, non-oxidized click conjugated alginate hydrogel retainedat least about 95% of the liposome load after 50 days, while Ca²⁺cross-linked alginate hydrogel retained about 85% of the liposome loadafter 40 days. This data demonstrates that, of the studied hydrogels,non-oxidized click conjugated alginate hydrogel is most effective atretaining liposomes over the period of 40 or more days.

FIG. 16c is a graph showing cumulative release (% load) of liposomesover the period of 28 days from Ca²⁺ cross-linked alginate and fromnon-oxidized click conjugated alginate hydrogels prepared at 5% w/valginate concentration. The liposome release profiles were measured invitro in PBS⁻⁻, which is a PBS buffer that does not contain Ca²⁺ or Mg²⁺ions.

The data in FIG. 16c indicates that about 60% of the liposome load isreleased from Ca²⁺ cross-linked alginate hydrogels after 28 days.Calcium cross-linking of alginate in the presence of liposomes leads tosignificant heterogeneity and sheer stress on encapsulated liposomes,resulting in liposome release. In contrast, non-oxidized clickconjugated alginate hydrogel is capable of retaining its liposome loadfor at least 20 days.

FIG. 16d is a graph showing cumulative release (% load) of liposomesfrom non-oxidized click conjugated alginate hydrogels and clickconjugated gelatin hydrogels over the period of 8 days. Addition ofalginate lyase capable of digesting alginate results in quick release ofliposomes from the non-oxidized click conjugated alginate hydrogels.Addition of collagenase capable of digesting gelatin results in quickrelease of liposomes from click conjugated gelatin hydrogels. Clickconjugated gelatin hydrogels are expected to be degraded in vivo becausecollagenases are ubiquitous in vivo, e.g., in a human body. In contrast,alginate lyases are not ubiquitous in vivo, therefore, alginatehydrogels are expected to remain intact in vivo if un-oxidized. Withoxidation, alginate degradation is pH dependent.

Example 17. Intactness of Liposomes after Encapsulation

The purpose of this experiment was to assess the ability of liposomesremain intact, e.g., maintain their diameter after encapsulation and berecovered from different hydrogels. The hydrogels studied in thisexperiment include calcium cross-linked alginate hydrogel, non-oxidizedclick conjugated alginate hydrogel and click conjugated gelatin hydrogelas described in Example 16. The size of the liposomes was measured bydynamic light scattering (DLS).

FIG. 17a is a dynamic light scattering (DLS) trace of a liposomestandard.

FIG. 17b is a DLS trace of a liposome which has been released from anon-oxidized click conjugated alginate hydrogel digested with alginatelyase after 8 days.

FIG. 17c is a DLS trace of a liposome which has been released after 28days from a non-oxidized click conjugated alginate hydrogel prepared atthe concentration of alginate of 5% w/v in PBS⁻ buffer.

FIG. 17d is a DLS trace of a liposome which has been released from aclick conjugated gelatin hydrogel digested with collagenase after 8days.

FIG. 17e is a DLS trace of a liposome which has been released after 3days from a calcium cross-linked alginate prepared in PBS⁻⁻ buffer.

FIG. 17f is a DLS trace of a liposome which has been released after 28days from a calcium cross-linked hydrogel prepared at the alginateconcentration of 5% w/v in PBS⁻ buffer.

The data in FIGS. 17a-17f demonstrate that liposomes released from clickconjugated alginate or gelatin hydrogels are of similar size as theliposome standard, which indicates that they remain intact after 28days. In contrast, the liposomes released from calcium cross-linkedalginate hydrogels have a substantially greater range in sizes ascompared to the liposome standard. This suggests that during the calciumcross-linking process the liposomes are torn apart due to sheer forcesand subsequently undergo confluence in solution, resulting in liposomalparticles of bigger size. Calcium cross-linking also introducessignificant heterogeneity due to poor distribution of calcium.

Example 18. Influence of the Degree of Alginate Oxidation on the Abilityof Alginate Hydrogels to Retain Liposomes

The purpose of this experiment was to assess the influence of the degreeof alginate oxidation in alginate hydrogels on their ability to retainencapsulated liposomes. This experiment utilized alginate hydrogels thatwere prepared from MVG alginate material that was cross-linked to Nb orTz using 250 equivalents of Nb or Tz in the click conjugation reaction.Prior to click conjugation, the alginate was oxidized to 0%, 5% or 10%total oxidation and then reductively processed with AB using theprocedures as described in Examples 1 and 2. Liposomes as described inExample 16 were encapsulated in the hydrogels, and their release wasmonitored in vitro by measuring the increase in Dil fluorescence in thesupernatant over the period of 75 days.

FIG. 18 is a graph showing cumulative release (% load) of liposomes over75 days from click conjugated alginate hydrogels that were oxidized to0%, 5% and 10% total oxidation and reductively processed with AB priorto click conjugation. The results in FIG. 18 demonstrate that oxidationof alginate to 5% or 10% and subsequent reduction of alginate prior toclick conjugation allows retention of liposomes over the period of 75days with detectable liposome release, while non-oxidized alginatereleases about 3% of liposomes over 75 days. Therefore, oxidation ofalginate improves the retention of liposomes by alginate hydrogels.

Example 19. Influence of pH on the Stability of Alginate Hydrogels

The purpose of this experiment was to investigate the release ofliposomal cargo from alginate hydrogels as a function of pH. Becausealginate biodegradation is acid or alkaline mediated, release ofliposomal cargo from alginate hydrogels was monitored at pH 5 (in 0.1 Msodium citrate buffer) and pH 9 (in 0.1 M sodium borate buffer) over 14days. Samples were prepared by firstly oxidizing MVG alginate to 20%oxidation, and subsequently reducing the material using ammonia borane.After 7 days, the pH 9 samples were mostly degraded, and after 14 days,the pH 5 samples were also found to be degraded.

FIG. 19a is a picture of tubes containing hydrogels with encapsulatedliposomes comprising 20% oxidized MVG that has been reduced with ammoniaborane, hydrogels and supernatants at day 0. FIG. 19b is a picture oftubes containing hydrogels with encapsulated liposomes, prepared using20% oxidized MVG that has been reduced with ammonia borane andsupernatants at day 1. FIG. 19c is a picture of tubes containinghydrogels with encapsulated liposomes, prepared using 20% oxidized MVG,that has been reduced with ammonia borane and supernatants at day 7.FIG. 19d is a picture of tubes containing hydrogels with encapsulatedliposomes, prepared using 20% oxidized MVG, that has been reduced withammonia borane and supernatants at day 14. FIG. 19e is a graph showingthe degradation based release of neutral liposomes (DOPC: Cholesterol)from alginate hydrogels that have been produced by oxidizing alginate to20% and subsequently reducing it with ammonia borane. Samples werereleased in MES buffer pH 6.5 to mimic the paratumoral microenvironment.

The results in FIGS. 19a-19d demonstrate that alginate is degraded andthe liposomes diffuse into the supernatant at the acidic pH (pH 5) orbasic pH (pH 9), but not at a neutral pH (7). Accordingly, liposomalrelease from click alginate hydrogels is pH sensitive and allows fordegradation mediated release of liposomal cargo.

Example 20. Encapsulation and Retention of Cationic Liposomes byAlginate Hydrogels

Previous experiments (e.g., the experiments described in Example 16)utilized DOPC/CHOL liposomes that were neutral. The purpose of thisexperiment was to investigate the ability of alginate hydrogels toencapsulate and retain charged liposomes e.g., cationic liposomes. Thisexperiment utilized cationic liposomes containingN-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate(DOTAP) and hydrogenated soy phosphatidilcholine (Hydro Soy PC) havingthe average diameter of 133 nm. The liposomes also contained thefluorescent dye 1,1′-Dioctadecyl-3,3,3′,3′-TetramethylindocarbocyaninePerchlorate (Dil). These liposomes were encapsulated in calciumcross-linked alginate material and in non-oxidized click conjugatedalginate that utilized 250 molar equivalents of click reagents in theconjugation reaction. Release of cationic liposomes from the hydrogelswas monitored as described in Example 16.

FIG. 20a is a DLS trace of a DOTAP: Hydro Soy PC liposome prior toextrusion, while FIG. 20b is a DLS trace of the same liposome afterextrusion. FIGS. 20a and 20b demonstrate that it is possible to preparecationic liposomes of a uniform size using extrusion.

FIG. 20c is a graph showing cumulative release (% load) of cationicliposomes over the period of 17 days from Ca²⁺ cross-linked alginate andfrom non-oxidized click conjugated alginate hydrogels. The results inFIG. 20c demonstrate that click conjugated alginate hydrogel canencapsulate and effectively retain cationic liposomes over the period of17 days. In contrast, release of cationic liposomes from the calciumcross-linked alginate hydrogel is evident after only 2 days.

Example 21. Protein Release Profiles from Algoxinol and AlgoxalateComposite Click Hydrogels

The goal of this experiment was to evaluate different protein releaseprofiles from the hydrogels prepared using different oxidized andreduced alginate materials that have been click conjugated. Thecompositions used to prepare the alginate hydrogels included: a) equalparts MVG at 10% w/v that has been oxidized to 50% and subsequentlyoxidized with sodium chlorite and conjugated with Tz at 2000 equivalentsand MVG at 10% w/v that has been oxidized to 10% and subsequentlyreduced with ammonia borane and conjugated with Nb at 2000 equivalents;b) equal parts MVG at 10% w/v that has been oxidized to 50% andsubsequently reduced with ammonia borane and conjugated with Tz at 250equivalents and MVG at 10% w/v that has been oxidized to 10% andsubsequently reduced with ammonia borane and conjugated with Nb at 250equivalents; c) 7 parts MVG at 10% w/v that has been oxidized to 10% andsubsequently reduced with ammonia borane and conjugated with Nb at 2000equivalents and 3 parts MVG at 10% w/v that has been oxidized to 50% andsubsequently reduced with ammonia borane and conjugated with Tz at 2000equivalents; and d) 3 parts MVG and 1 part MVG that has been crosslinkedwith calcium sulfate.

Protein release curves were evaluated by encapsulating Human IGF andhuman VEGF (R&D Systems) in various click hydrogels. Samples wereprepared by first separately dissolving freeze-dried Alg-N and Alg-Tpolymers of various degrees of substitution to final desiredconcentration (w/v) in PBS. The proteins were added and mixed with theAlg-N solution to reach a 2 μg/gel (100 μL) final concentration. TheProtein-Alg-N solution was thoroughly mixed with the Alg-T solution andwas allowed to gel for at least 30 minutes. Samples were created byadding 1 mL of PBS with 1% BSA to each gel and incubated at 37° C.Samples were collected at various timepoints with replacement of thesupernatant at each timepoint. Supernatant protein content with VEGF andIGF was quantified with Human VEGF and IGF Quantikine ELISA kits (R&DSystems) using a standard curve of comparable sample matrix (PBS w/1%BSA and PBS w/5% Tween 20, respectively).

FIG. 21a is a graph showing the release of IGF-1 from click conjugatedalginate hydrogels prepared using various alginate compositions. FIG.21b is a graph showing the release of VEGF₁₆₅ from click alginatehydrogels prepared using various alginate compositions.

The data in FIGS. 21a and 21b show the ability to modulate the burstrelease of IGF-1 and VEGF₁₆₅ using the click gels prepared usingdifferent alginate compositions. In contrast, calcium crosslinkedalginate hydrogels are unable to modulate diffusion. This exampledemonstrates that it is possible to modulate protein release via variouscombinations of the algoxinol and algoxalate containing alginate,alginate degree of substitution, alginate concentration, and ratio of Nbto Tz.

Example 22. Viscosity of Alginate Solutions

The goal of this experiment was to investigate the viscosity of clickconjugated alginate solutions as a function of alginate oxidation and/orreduction. To this end, click alginate solutions were evaluated forrelative viscosity by first separately dissolving freeze-dried Alg-N andAlg-T polymers to final desired concentration (w/v) in ddH₂O. Solutionswere directly pipetted onto the bottom plate of a TA Instruments ARG2rheometer equipped with 20 mm flat upper plate geometry and a 400-microngeometry gap. A Peltier cooler was used to control the temperature (20°C.). Alginate samples were subjected to strains between 0.1 and 1 Hz.

FIG. 22 is a bar graph showing the relative viscosities of variousalginate solutions at different alginate concentrations (% w/v), degreesof oxidation, and processing with either sodium chlorite or ammoniaborane. The data demonstrate that click conjugation to MVG withoutoxidation does not result in substantial differences in viscosity,whereas the viscosity is substantially reduced with click conjugation toalginate containing either algoxinol or algoxalate, and is comparable tothat of water (0.890 cPa at 25° C.).

What is claimed is:
 1. A composition comprising a highly oxidizedpolysaccharide and a cross-linking agent attached to the highly oxidizedpolysaccharide.
 2. The composition of claim 1, wherein said highlyoxidized polysaccharide is an alginate polymer.
 3. The composition ofclaim 2, wherein said highly oxidized polysaccharide comprisesalgoxalate having the following structure:


4. The composition of claim 1, wherein said highly oxidizedpolysaccharide is produced by a method comprising the steps of: reactingthe polysaccharide with a diol specific oxidizing agent to produce analdehyde containing oxidized polysaccharide; and reacting said oxidizedpolysaccharide with a second oxidizing agent which converts aldehydesinto carboxylic acids to produce said highly oxidized polysaccharide. 5.The composition of claim 4, wherein said diol specific oxidizing agentis sodium periodate.
 6. The composition of claim 4, wherein said secondoxidizing agent is sodium chlorite.
 7. The composition of claim 1,wherein said cross-linking agent is a click reagent.
 8. The compositionof claim 7, wherein said click reagent is selected from the groupconsisting of azide, dibenzocyclooctyne (DBCO), transcyclooctene,tetrazine and norbornene and variants thereof.
 9. A hydrogel comprisingthe composition of claim
 1. 10. The hydrogel of claim 9, furthercomprising a therapeutic agent.
 11. The hydrogel of claim 10, whereinsaid therapeutic agent is selected from the group consisting of a cell,a small molecule and a biologic.
 12. A hydrogel comprising a pluralityof highly oxidized polysaccharides cross-linked to each other, whereinsaid hydrogel comprises a mesh size of 10 nm or less.
 13. The hydrogelof claim 12, wherein said highly oxidized polysaccharides are highlyoxidized alginate polymers.
 14. The hydrogel of claim 13, wherein saidhighly oxidized alginate polymers are produced by a method comprisingthe steps of: reacting an alginate with a diol specific oxidizing agentto produce an aldehyde containing oxidized alginate; and reacting saidaldehyde containing oxidized alginate with a second oxidizing agentwhich converts aldehydes into carboxylic acids, thereby producing saidhighly oxidized alginate polymers.
 15. The hydrogel of claim 14, whereinsaid diol specific oxidizing agent is sodium periodate.
 16. The hydrogelof claim 14, wherein said second oxidizing agent is sodium chlorite. 17.The hydrogel of claim 13, wherein said highly oxidized alginate polymersare about 0.1% 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% oxidized.
 18. The hydrogelof claim 12, further comprising a therapeutic or diagnostic agent. 19.The hydrogel of claim 18, wherein said therapeutic agent is selectedfrom the group consisting of a cell, a small molecule and a biologic.20. The hydrogel of claim 12, wherein said polysaccharides comprise across-linking agent attached to the polysaccharides.
 21. The hydrogel ofclaim 20, wherein said cross-linking agent is a click reagent.
 22. Thehydrogel of claim 21, wherein said click reagent is selected from thegroup consisting of azide, dibenzocyclooctyne, transcyclooctene,tetrazine and norbornene and variants thereof.
 23. An implantable orinjectable device comprising the hydrogel of claim
 12. 24. A drugdelivery composition comprising a lipid based nanoparticle encapsulatinga therapeutic or diagnostic agent; and the hydrogel of claim 12encapsulating said lipid based nanoparticle.
 25. A method for treating asubject in need thereof, the method comprising administering to saidsubject an effective amount of the hydrogel of claim 12, therebytreating said subject.
 26. A method of treating chronic ischemia orenhancing engraftment of a transplanted tissue in a subject in needthereof, the method comprising administering to said subject aneffective amount of the hydrogel of claim 12, thereby treating saidchronic ischemia or enhancing engraftment of said transplanted tissue insaid subject.